De novo synthesized gene libraries

ABSTRACT

De novo synthesized large libraries of nucleic acids are provided herein with low error rates. Further, devices for the manufacturing of high-quality building blocks, such as oligonucleotides, are described herein. Longer nucleic acids can be synthesized in parallel using microfluidic assemblies. Further, methods herein allow for the fast construction of large libraries of long, high-quality genes. Devices for the manufacturing of large libraries of long and high-quality nucleic acids are further described herein.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.15/233,835 filed on Aug. 10, 2016, which is a divisional of U.S.application Ser. No. 15/187,714 filed on Jun. 20, 2016, which is acontinuation of U.S. application Ser. No. 14/452,429, filed Aug. 5,2014, now U.S. Pat. No. 9,409,139 issued Aug. 9, 2016, which claims thebenefit of U.S. Provisional Application No. 61/862,445, filed Aug. 5,2013 and U.S. Provisional Application No. 61/862,457, filed Aug. 5,2013, which applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 29, 2014 is44854-701_309_SL.txt and is 30,766 bytes in size.

BACKGROUND OF THE INVENTION

Highly efficient chemical gene synthesis with high fidelity and low costhas a central role in biotechnology and medicine, and in basicbiomedical research.

De novo gene synthesis is a powerful tool for basic biological researchand biotechnology applications. While various methods are known for thesynthesis of relatively short fragments in a small scale, thesetechniques suffer from scalability, automation, speed, accuracy, andcost. There is a need for devices for simple, reproducible, scalable,less error-prone and cost-effective methods that guarantee successfulsynthesis of desired genes and are amenable to automation.

SUMMARY OF THE INVENTION

As noted above, there exists a pressing need for methods, devices andsystems that can quickly synthesize large gene libraries or relativelylonger oligonucleotide fragments efficiently with less error. Similarly,there is also a need for methods that can partition and mix liquidreagents in a microfluidic scale for large numbers of individuallyaddressable reactions in parallel. The present invention addresses theseneeds and provides related advantages as well.

In one aspect, the present invention provides a gene library asdescribed herein. The gene library comprises a collection of genes. Insome embodiments, the collection comprises at least 100 differentpreselected synthetic genes that can be of at least 0.5 kb length withan error rate of less than 1 in 3000 bp compared to predeterminedsequences comprising the genes. In another aspect, the present inventionalso provides a gene library that comprises a collection of genes. Thecollection may comprise at least 100 different preselected syntheticgenes that can be each of at least 0.5 kb length. At least 90% of thepreselected synthetic genes may comprise an error rate of less than 1 in3000 bp compared to predetermined sequences comprising the genes.Desired predetermined sequences may be supplied by any method, typicallyby a user, e.g. a user entering data using a computerized system. Invarious embodiments, synthesized nucleic acids are compared againstthese predetermined sequences, in some cases by sequencing at least aportion of the synthesized nucleic acids, e.g. using next-generationsequencing methods. In some embodiments related to any of the genelibraries described herein, at least 90% of the preselected syntheticgenes comprise an error rate of less than 1 in 5000 bp compared topredetermined sequences comprising the genes. In some embodiments, atleast 0.05% of the preselected synthetic genes are error free. In someembodiments, at least 0.5% of the preselected synthetic genes are errorfree. In some embodiments, at least 90% of the preselected syntheticgenes comprise an error rate of less than 1 in 3000 bp compared topredetermined sequences comprising the genes. In some embodiments, atleast 90% of the preselected synthetic genes are error free orsubstantially error free. In some embodiments, the preselected syntheticgenes comprise a deletion rate of less than 1 in 3000 bp compared topredetermined sequences comprising the genes. In some embodiments, thepreselected synthetic genes comprise an insertion rate of less than 1 in3000 bp compared to predetermined sequences comprising the genes. Insome embodiments, the preselected synthetic genes comprise asubstitution rate of less than 1 in 3000 bp compared to predeterminedsequences comprising the genes. In some embodiments, the gene library asdescribed herein further comprises at least 10 copies of each syntheticgene. In some embodiments, the gene library as described herein furthercomprises at least 100 copies of each synthetic gene. In someembodiments, the gene library as described herein further comprises atleast 1000 copies of each synthetic gene. In some embodiments, the genelibrary as described herein further comprises at least 1000000 copies ofeach synthetic gene. In some embodiments, the collection of genes asdescribed herein comprises at least 500 genes. In some embodiments, thecollection comprises at least 5000 genes. In some embodiments, thecollection comprises at least 10000 genes. In some embodiments, thepreselected synthetic genes are at least 1 kb. In some embodiments, thepreselected synthetic genes are at least 2 kb. In some embodiments, thepreselected synthetic genes are at least 3 kb. In some embodiments, thepredetermined sequences comprise less than 20 bp in addition compared tothe preselected synthetic genes. In some embodiments, the predeterminedsequences comprise less than 15 bp in addition compared to thepreselected synthetic genes. In some embodiments, at least one of thesynthetic genes differs from any other synthetic gene by at least 0.1%.In some embodiments, each of the synthetic genes differs from any othersynthetic gene by at least 0.1%. In some embodiments, at least one ofthe synthetic genes differs from any other synthetic gene by at least10%. In some embodiments, each of the synthetic genes differs from anyother synthetic gene by at least 10%. In some embodiments, at least oneof the synthetic genes differs from any other synthetic gene by at least2 base pairs. In some embodiments, each of the synthetic genes differsfrom any other synthetic gene by at least 2 base pairs. In someembodiments, the gene library as described herein further comprisessynthetic genes that are of less than 2 kb with an error rate of lessthan 1 in 20000 bp compared to preselected sequences of the genes. Insome embodiments, a subset of the deliverable genes is covalently linkedtogether. In some embodiments, a first subset of the collection of genesencodes for components of a first metabolic pathway with one or moremetabolic end products. In some embodiments, the gene library asdescribed herein further comprises selecting of the one or moremetabolic end products, thereby constructing the collection of genes. Insome embodiments, the one or more metabolic end products comprise abiofuel. In some embodiments, a second subset of the collection of genesencodes for components of a second metabolic pathway with one or moremetabolic end products. In some embodiments, the gene library is in aspace that is less than 100 m³. In some embodiments, the gene library isin a space that is less than 1 m³. In some embodiments, the gene libraryis in a space that is less than 1 m³.

In another aspect, the present invention also provides a method ofconstructing a gene library. The method comprises the steps of: enteringbefore a first timepoint, in a computer readable non-transient medium atleast a first list of genes and a second list of genes, wherein thegenes are at least 500 bp and when compiled into a joint list, the jointlist comprises at least 100 genes; synthesizing more than 90% of thegenes in the joint list before a second timepoint, thereby constructinga gene library with deliverable genes. In some embodiments, the secondtimepoint is less than a month apart from the first timepoint.

In practicing any of the methods of constructing a gene library asprovided herein, the method as described herein further comprisesdelivering at least one gene at a second timepoint. In some embodiments,at least one of the genes differs from any other gene by at least 0.1%in the gene library. In some embodiments, each of the genes differs fromany other gene by at least 0.1% in the gene library. In someembodiments, at least one of the genes differs from any other gene by atleast 10% in the gene library. In some embodiments, each of the genesdiffers from any other gene by at least 10% in the gene library. In someembodiments, at least one of the genes differs from any other gene by atleast 2 base pairs in the gene library. In some embodiments, each of thegenes differs from any other gene by at least 2 base pairs in the genelibrary. In some embodiments, at least 90% of the deliverable genes areerror free. In some embodiments, the deliverable genes comprises anerror rate of less than 1/3000 resulting in the generation of a sequencethat deviates from the sequence of a gene in the joint list of genes. Insome embodiments, at least 90% of the deliverable genes comprise anerror rate of less than 1 in 3000 bp resulting in the generation of asequence that deviates from the sequence of a gene in the joint list ofgenes. In some embodiments, genes in a subset of the deliverable genesare covalently linked together. In some embodiments, a first subset ofthe joint list of genes encode for components of a first metabolicpathway with one or more metabolic end products. In some embodiments,any of the methods of constructing a gene library as described hereinfurther comprises selecting of the one or more metabolic end products,thereby constructing the first, the second or the joint list of genes.In some embodiments, the one or more metabolic end products comprise abiofuel. In some embodiments, a second subset of the joint list of genesencode for components of a second metabolic pathway with one or moremetabolic end products. In some embodiments, the joint list of genescomprises at least 500 genes. In some embodiments, the joint list ofgenes comprises at least 5000 genes. In some embodiments, the joint listof genes comprises at least 10000 genes. In some embodiments, the genescan be at least 1 kb. In some embodiments, the genes are at least 2 kb.In some embodiments, the genes are at least 3 kb. In some embodiments,the second timepoint is less than 25 days apart from the firsttimepoint. In some embodiments, the second timepoint is less than 5 daysapart from the first timepoint. In some embodiments, the secondtimepoint is less than 2 days apart from the first timepoint. It isnoted that any of the embodiments described herein can be combined withany of the methods, devices or systems provided in the currentinvention.

In another aspect, a method of constructing a gene library is providedherein. The method comprises the steps of: entering at a firsttimepoint, in a computer readable non-transient medium a list of genes;synthesizing more than 90% of the list of genes, thereby constructing agene library with deliverable genes; and delivering the deliverablegenes at a second timepoint. In some embodiments, the list comprises atleast 100 genes and the genes can be at least 500 bp. In still yet someembodiments, the second timepoint is less than a month apart from thefirst timepoint.

In practicing any of the methods of constructing a gene library asprovided herein, in some embodiments, the method as described hereinfurther comprises delivering at least one gene at a second timepoint. Insome embodiments, at least one of the genes differs from any other geneby at least 0.1% in the gene library. In some embodiments, each of thegenes differs from any other gene by at least 0.1% in the gene library.In some embodiments, at least one of the genes differs from any othergene by at least 10% in the gene library. In some embodiments, each ofthe genes differs from any other gene by at least 10% in the genelibrary. In some embodiments, at least one of the genes differs from anyother gene by at least 2 base pairs in the gene library. In someembodiments, each of the genes differs from any other gene by at least 2base pairs in the gene library. In some embodiments, at least 90% of thedeliverable genes are error free. In some embodiments, the deliverablegenes comprises an error rate of less than 1/3000 resulting in thegeneration of a sequence that deviates from the sequence of a gene inthe list of genes. In some embodiments, at least 90% of the deliverablegenes comprise an error rate of less than 1 in 3000 bp resulting in thegeneration of a sequence that deviates from the sequence of a gene inthe list of genes. In some embodiments, genes in a subset of thedeliverable genes are covalently linked together. In some embodiments, afirst subset of the list of genes encode for components of a firstmetabolic pathway with one or more metabolic end products. In someembodiments, the method of constructing a gene library further comprisesselecting of the one or more metabolic end products, therebyconstructing the list of genes. In some embodiments, the one or moremetabolic end products comprise a biofuel. In some embodiments, a secondsubset of the list of genes encode for components of a second metabolicpathway with one or more metabolic end products. It is noted that any ofthe embodiments described herein can be combined with any of themethods, devices or systems provided in the current invention.

In practicing any of the methods of constructing a gene library asprovided herein, in some embodiments, the list of genes comprises atleast 500 genes. In some embodiments, the list comprises at least 5000genes. In some embodiments, the list comprises at least 10000 genes. Insome embodiments, the genes are at least 1 kb. In some embodiments, thegenes are at least 2 kb. In some embodiments, the genes are at least 3kb. In some embodiments, the second timepoint as described in themethods of constructing a gene library is less than 25 days apart fromthe first timepoint. In some embodiments, the second timepoint is lessthan 5 days apart from the first timepoint. In some embodiments, thesecond timepoint is less than 2 days apart from the first timepoint. Itis noted that any of the embodiments described herein can be combinedwith any of the methods, devices or systems provided in the currentinvention.

In another aspect, the present invention also provides a method ofsynthesizing n-mer oligonucleotides on a substrate. The method comprisesa) providing a substrate with resolved loci that are functionalized witha chemical moiety suitable for nucleotide coupling; and b) coupling atleast two building blocks to a plurality of growing oligonucleotidechains each residing on one of the resolved loci at a rate of at least12 nucleotides per hour according to a locus specific predeterminedsequence, thereby synthesizing a plurality of oligonucleotides that aren basepairs long. Various embodiments related to the method ofsynthesizing n-mer oligonucleotides on a substrate are described herein.

In any of the methods of synthesizing n-mer oligonucleotides on asubstrate as provided herein, in some embodiments, the methods furthercomprise coupling at least two building blocks to a plurality of growingoligonucleotide chains each residing on one of the resolved loci at arate of at least 15 nucleotides per hour. In some embodiments, themethod further comprises coupling at least two building blocks to aplurality of growing oligonucleotide chains each residing on one of theresolved loci at a rate of at least 20 nucleotides per hour. In someembodiments, the method further comprises coupling at least two buildingblocks to a plurality of growing oligonucleotide chains each residing onone of the resolved loci at a rate of at least 25 nucleotides per hour.In some embodiments, at least one resolved locus comprises n-meroligonucleotides deviating from the locus specific predeterminedsequence with an error rate of less than 1/500 bp. In some embodiments,at least one resolved locus comprises n-mer oligonucleotides deviatingfrom the locus specific predetermined sequence with an error rate ofless than 1/1000 bp. In some embodiments, at least one resolved locuscomprises n-mer oligonucleotides deviating from the locus specificpredetermined sequence with an error rate of less than 1/2000 bp. Insome embodiments, the plurality of oligonucleotides on the substratedeviate from respective locus specific predetermined sequences at anerror rate of less than 1/500 bp. In some embodiments, the plurality ofoligonucleotides on the substrate deviate from respective locus specificpredetermined sequences at an error rate of less than 1/1000 bp. In someembodiments, the plurality of oligonucleotides on the substrate deviatefrom respective locus specific predetermined sequences at an error rateof less than 1/2000 bp.

In practicing any of the methods of synthesizing n-mer oligonucleotideson a substrate as provided herein, in some embodiments, the buildingblocks comprise an adenine, guanine, thymine, cytosine, or uridinegroup, or a modified nucleotide. In some embodiments, the buildingblocks comprise a modified nucleotide. In some embodiments, the buildingblocks comprise dinucleotides or trinucleotides. In some embodiments,the building blocks comprise phosphoramidite. In some embodiments, n ofthe n-mer oligonucleotides is at least 100. In some embodiments, n is atleast 200. In some embodiments, n is at least 300. In some embodiments,n is at least 400. In some embodiments, the surface comprises at least100,000 resolved loci and at least two of the plurality of growingoligonucleotides can be different from each other.

In some embodiments, the method of synthesizing n-mer oligonucleotideson a substrate as described herein further comprises vacuum drying thesubstrate before coupling. In some embodiments, the building blockscomprise a blocking group. In some embodiments, the blocking groupcomprises an acid-labile DMT. In some embodiments, the acid-labile DMTcomprises 4,4′-dimethoxytrityl. In some embodiments, the method ofsynthesizing n-mer oligonucleotides on a substrate as described hereinfurther comprises oxidation or sulfurization. In some embodiments, themethod of synthesizing n-mer oligonucleotides on a substrate asdescribed herein further comprises chemically capping uncoupledoligonucleotide chains. In some embodiments, the method of synthesizingn-mer oligonucleotides on a substrate as described herein furthercomprises removing the blocking group, thereby deblocking the growingoligonucleotide chain. In some embodiments, the position of thesubstrate during the coupling step is within 10 cm of the position ofthe substrate during the vacuum drying step. In some embodiments, theposition of the substrate during the coupling step is within 10 cm ofthe position of the substrate during the oxidation step. In someembodiments, the position of the substrate during the coupling step iswithin 10 cm of the position of the substrate during the capping step.In some embodiments, the position of the substrate during the couplingstep is within 10 cm of the position of the substrate during thedeblocking step. In some embodiments, the substrate comprises at least10,000 vias providing fluid communication between a first surface of thesubstrate and a second surface of the substrate. In some embodiments,the substrate comprises at least 100,000 vias providing fluidcommunication between a first surface of the substrate and a secondsurface of the substrate. In some embodiments, the substrate comprisesat least 1,000,000 vias providing fluid communication between a firstsurface of the substrate and a second surface of the substrate. It isnoted that any of the embodiments described herein can be combined withany of the methods, devices or systems provided in the currentinvention.

In another aspect of the present invention, a system for conducting aset of parallel reactions is provided herein. The system comprises: afirst surface with a plurality of resolved loci; a capping element witha plurality of resolved reactor caps. In some embodiments, the systemaligns the plurality of resolved reactor caps with the plurality ofresolved loci on the first surface forming a temporary seal between thefirst surface and the capping element, thereby physically dividing theloci on the first surface into groups of at least two loci into areactor associated with each reactor cap. In some embodiments, eachreactor holds a first set of reagents.

In some embodiments related to any of the systems for conducting a setof parallel reactions as described herein, upon release from the firstsurface, the reactor caps retain at least a portion of the first set ofreagents. In some embodiments, the portion is about 30%. In someembodiments, the portion is about 90%. In some embodiments, theplurality of resolved loci resides on microstructures fabricated into asupport surface. In some embodiments, the plurality of resolved loci isat a density of at least 1 per mm². In some embodiments, the pluralityof resolved loci is at a density of at least 10 per mm². In someembodiments, the plurality of resolved loci are at a density of at least100 per mm². In some embodiments, the microstructures comprise at leasttwo channels in fluidic communication with each other. In someembodiments, the at least two channels comprise two channels withdifferent width. In some embodiments, at least two channels comprise twochannels with different length. In some embodiments, at least one of thechannels is longer than 100 μm. In some embodiments, at least one of thechannels is shorter than 1000 μm. In some embodiments, at least one ofthe channels is wider than 50 μm in diameter. In some embodiments, atleast one of the channels is narrower than 100 μm in diameter. In someembodiments, the system further comprises a second surface with aplurality of resolved loci at a density of at least 0.1 per mm². In someembodiments, the system further comprises a second surface with aplurality of resolved loci at a density of at least 1 per mm². In someembodiments, the system further comprises a second surface with aplurality of resolved loci at a density of at least 10 per mm².

In some embodiments related to any of the systems for conducting a setof parallel reactions as described herein, the resolved loci of thefirst surface comprise a coating of reagents. In some embodiments, theresolved loci of the second surface comprise a coating of reagents. Insome embodiments, the coating of reagents is covalently linked to thefirst or second surface. In some embodiments, the coating of reagentscomprises oligonucleotides. In some embodiments, the coating of reagentshas a surface area of at least 1.45 μm² per 1.0 μm² of planar surfacearea. In some embodiments, the coating of reagents has a surface area ofat least 1.25 μm² per 1.0 μm² of planar surface area. In someembodiments, the coating of reagents has a surface area of at least 1μm² per 1.0 μm² of planar surface area. In some embodiments, theresolved loci in the plurality of resolved loci comprise a nominalarclength of the perimeter at a density of at least 0.001 μm/μm². Insome embodiments, the resolved loci in the plurality of resolved locicomprise a nominal arclength of the perimeter at a density of at least0.01 μm/μm². In some embodiments, the resolved loci in the plurality ofresolved loci of the first surface comprise a high energy surface. Insome embodiments, the first and second surfaces comprise a differentsurface tension with a given liquid. In some embodiments, the highsurface energy corresponds to a water contact angle of less than 20degree. In some embodiments, the plurality of resolved loci are locatedon a solid substrate comprising a material selected from the groupconsisting of silicon, polystyrene, agarose, dextran, cellulosicpolymers, polyacrylamides, PDMS, and glass. In some embodiments, thecapping elements comprise a material selected from the group consistingof silicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, PDMS, and glass. It is noted that any of theembodiments described herein can be combined with any of the methods,devices or systems provided in the current invention.

In yet another aspect, the present invention also provides an array ofenclosures. The array of enclosures comprise: a plurality of resolvedreactors comprising a first substrate and a second substrate comprisingreactor caps; at least 2 resolved loci in each reactor. In some cases,the resolved reactors are separated with a releasable seal. In somecases, the reactor caps retain at least a part of the contents of thereactors upon release of the second substrate from the first substrate.In some embodiments, the reactor caps on the second substrate have adensity of at least 0.1 per mm². In some embodiments, reactor caps onthe second substrate have a density of at least 1 per mm². In someembodiments, reactor caps on the second substrate have a density of atleast 10 per mm².

In some embodiments related to the array of enclosures as providedherein, the reactor caps retain at least 30% of the contents of thereactors. In some embodiments, the reactor caps retain at least 90% ofthe contents of the reactors. In some embodiments, the resolved loci areat a density of at least 2/mm². In some embodiments, the resolved lociare at a density of at least 100/mm². In some embodiments, the array ofenclosures further comprises at least 5 resolved loci in each reactor.In some embodiments, the array of enclosures as described herein furthercomprises at least 20 resolved loci in each reactor. In someembodiments, the array of enclosures as described herein furthercomprises at least 50 resolved loci in each reactor. In someembodiments, the array of enclosures as described herein furthercomprises at least 100 resolved loci in each reactor.

In some embodiments related to the array of enclosures as describedherein, the resolved loci reside on microstructures fabricated into asupport surface. In some embodiments, the microstructures comprise atleast two channels in fluidic communication with each other. In someembodiments, the at least two channels comprise two channels withdifferent width. In some embodiments, the at least two channels comprisetwo channels with different length. In some embodiments, at least one ofthe channels is longer than 100 μm. In some embodiments, at least one ofthe channels is shorter than 1000 μm. In some embodiments, at least oneof the channels is wider than 50 μm in diameter. In some embodiments, atleast one of the channels is narrower than 100 μm in diameter. In someembodiments, the microstructures comprise a nominal arclength of theperimeter of the at least two channels that has a density of at least0.01 μm/square μm. In some embodiments, the microstructures comprise anominal arclength of the perimeter of the at least two channels that hasa density of at least 0.001 μm/square μm. In some embodiments, theresolved reactors are separated with a releasable seal. In someembodiments, the seal comprises a capillary burst valve.

In some embodiments related to the array of enclosures as describedherein, the plurality of resolved loci of the first substrate comprise acoating of reagents. In some embodiments, the plurality of resolved lociof the second substrate comprises a coating of reagents. In someembodiments, the coating of reagents is covalently linked to the firstor second surface. In some embodiments, the coating of reagentscomprises oligonucleotides. In some embodiments, the coating of reagentshas a surface area of at least 1 μm² per 1.0 μm² of planar surface area.In some embodiments, the coating of reagents has a surface area of atleast 1.25 μm² per 1.0 μm² of planar surface area. In some embodiments,the coating of reagents has a surface area of at least 1.45 μm² per 1.0μm² of planar surface area. In some embodiments, the plurality ofresolved loci of the first substrate comprises a high energy surface. Insome embodiments, the first and second substrates comprise a differentsurface tension with a given liquid. In some embodiments, the surfaceenergy corresponds to a water contact angle of less than 20 degree. Insome embodiments, the plurality of resolved loci or the reactor caps arelocated on a solid substrate comprising a material selected from thegroup consisting of silicon, polystyrene, agarose, dextran, cellulosicpolymers, polyacrylamides, PDMS, and glass. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays or systems provided in the current invention.

In still yet another aspect, the present invention also provides amethod of conducting a set of parallel reactions. The method comprises:(a) providing a first surface with a plurality of resolved loci; (b)providing a capping element with a plurality of resolved reactor caps;(c) aligning the plurality of resolved reactor caps with the pluralityof resolved loci on the first surface and forming a temporary sealbetween the first surface and the capping element, thereby physicallydividing the loci on the first surface into groups of at least two loci;(d) performing a first reaction, thereby forming a first set ofreagents; and (e) releasing the capping element from the first surface,wherein each reactor cap retains at least a portion of the first set ofreagents in a first reaction volume. In some embodiments, the portion isabout 30%. In some embodiments, the portion is about 90%.

In some embodiments, the method of conducting a set of parallelreactions as described herein further comprises the steps of: (f)providing a second surface with a plurality of resolved loci; (g)aligning the plurality of resolved reactor caps with the plurality ofresolved loci on the second surface and forming a temporary seal betweenthe second surface and the capping element, thereby physically dividingthe loci on the second surface; (h) performing a second reaction usingthe portion of the first set of reagents, thereby forming a second setof reagents; and (i) releasing the capping element from the secondsurface, wherein each reactor cap can retain at least a portion of thesecond set of reagents in a second reaction volume. In some embodiments,the portion is about 30%. In some embodiments, the portion is about 90%.

In practicing any of the methods of conducting a set of parallelreactions as described herein, the plurality of resolved loci can have adensity of at least 1 per mm² on the first surface. In some embodiments,the plurality of resolved loci have a density of at least 10 per mm² onthe first surface. In some embodiments, the plurality of resolved locihave a density of at least 100 per mm² on the first surface. In someembodiments, the plurality of resolved reactor caps have a density of atleast 0.1 per mm² on the capping element. In some embodiments, theplurality of resolved reactor caps have a density of at least 1 per mm²on the capping element. In some embodiments, the plurality of resolvedreactor caps have a density of at least 10 per mm² on the cappingelement. In some embodiments, the plurality of resolved loci have adensity of more than 0.1 per mm² on the second surface. In someembodiments, the plurality of resolved loci have a density of more than1 per mm² on the second surface. In some embodiments, the plurality ofresolved loci have a density of more than 10 per mm² on the secondsurface.

In practicing any of the methods of conducting a set of parallelreactions as described herein, the releasing of the capping elementsfrom the surface steps such as the releasing steps in (e) and (i) asdescribed herein can be performed at a different velocity. In someembodiments, the resolved loci of the first surface comprise a coatingof reagents for the first reaction. In some embodiments, the resolvedloci of the second surface comprise a coating of reagents for the secondreaction. In some embodiments, the coating of reagents is covalentlylinked to the first or second surface. In some embodiments, the coatingof reagents comprises oligonucleotides. In some embodiments, the coatingof reagents has a surface area of at least 1 μm² per 1.0 μm² of planarsurface area. In some embodiments, the coating of reagents has a surfacearea of at least 1.25 μm² per 1.0 μm² of planar surface area. In someembodiments, the coating of reagents has a surface area of at least 1.45μm² per 1.0 μm² of planar surface area. In some embodiments, theoligonucleotides are at least 25 bp. In some embodiments, theoligonucleotides are at least 200 bp. In some embodiments, theoligonucleotides are at least 300 bp. In some embodiments, the resolvedloci of the first surface comprise a high energy surface. In someembodiments, the first and second surfaces comprise a different surfacetension with a given liquid. In some embodiments, the surface energycorresponds to a water contact angle of less than 20 degree.

In some embodiments related to the method of conducting a set ofparallel reactions as described herein, the plurality of resolved locior the resolved reactor caps are located on a solid substrate comprisinga material selected from the group consisting of silicon, polystyrene,agarose, dextran, cellulosic polymers, polyacrylamides, PDMS, and glass.In some embodiments, the first and second reaction volumes aredifferent. In some embodiments, the first or second reaction comprisespolymerase cycling assembly. In some embodiments, the first or secondreaction comprises enzymatic gene synthesis, annealing and ligationreaction, simultaneous synthesis of two genes via a hybrid gene, shotgunligation and co-ligation, insertion gene synthesis, gene synthesis viaone strand of DNA, template-directed ligation, ligase chain reaction,microarray-mediated gene synthesis, solid-phase assembly, Sloningbuilding block technology, or RNA ligation mediated gene synthesis. Insome embodiments, the methods of conducting a set of parallel reactionsas described herein further comprises cooling the capping element. Insome embodiments, the method of conducting a set of parallel reactionsas described herein further comprises cooling the first surface. In someembodiments, the method of conducting a set of parallel reactions asdescribed herein further comprises cooling the second surface. It isnoted that any of the embodiments described herein can be combined withany of the methods, devices, arrays or systems provided in the currentinvention.

In another aspect, the present invention provides a substrate having afunctionalized surface. The substrate having a functionalized surfacecan comprise a solid support having a plurality of resolved loci. Insome embodiments, the resolved loci are functionalized with a moietythat increases the surface energy of the solid support. In someembodiments, the resolved loci are localized on microchannels.

In some embodiments related to the substrate having a functionalizedsurface as described herein, the moiety is a chemically inert moiety. Insome embodiments, the microchannels comprise a volume of less than 1 nl.In some embodiments, the microchannels comprise a density of the nominalarclength of the perimeter of 0.036 μm/square μm. In some embodiments,the functionalized surface comprises a nominal surface area of at least1 μm² per 1.0 μm² of planar surface area of the substrate. In someembodiments, the functionalized surface comprises a nominal surface areaof at least 1.25 μm² per 1.0 μm² of planar surface area of thesubstrate. In some embodiments, the functionalized surface comprises anominal surface area of at least 1.45 μm² per 1.0 μm² of planar surfacearea of the substrate. In some embodiments, the resolved loci in theplurality of resolved loci comprise a coating of reagents. In someembodiments, the coating of reagents is covalently linked to thesubstrate. In some embodiments, the coating of reagents comprisesoligonucleotides. In some embodiments, at least one of the microchannelsis longer than 100 μm. In some embodiments, at least one of themicrochannels is shorter than 1000 μm. In some embodiments, at least oneof the microchannels is wider than 50 μm in diameter. In someembodiments, at least one of the microchannels is narrower than 100 μmin diameter. In some embodiments, the surface energy corresponds to awater contact angle of less than 20 degree. In some embodiments, thesolid support comprises a material selected from the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, PDMS, and glass. In some embodiments, the plurality ofresolved loci is at a density of at least 1/mm². In some embodiments,the plurality of resolved loci is at a density of at least 100/mm². Itis noted that any of the embodiments described herein can be combinedwith any of the methods, devices, arrays, substrates or systems providedin the current invention.

In another aspect, the present invention also provides a method forsynthesizing oligonucleotides on a substrate having a functionalizedsurface. The method comprises: (a) applying through at least one inkjetpump at least one drop of a first reagent to a first locus of aplurality of loci; (b) applying negative pressure to the substrate; and(c) applying through at least one inkjet pump at least one drop of asecond reagent to the first locus.

In practicing any of the methods for synthesizing oligonucleotides on asubstrate having a functionalized surface as described herein, the firstand second reagents can be different. In some embodiments, the firstlocus is functionalized with a moiety that increases their surfaceenergy. In some embodiments, the moiety is a chemically inert moiety. Insome embodiments, the plurality of loci resides on microstructuresfabricated into the substrate surface. In some embodiments, themicrostructures comprise at least two channels in fluidic communicationwith each other. In some embodiments, the at least two channels comprisetwo channels with different width. In some embodiments, the at least twochannels comprise two channels with different length. In someembodiments, at least one of the channels is longer than 100 μm. In someembodiments, at least one of the channels is shorter than 1000 μm. Insome embodiments, at least one of the channels is wider than 50 μm indiameter. In some embodiments, at least one of the channels is narrowerthan 100 μm in diameter. In some embodiments, the substrate surfacecomprises a material selected from the group consisting of silicon,polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides,PDMS, and glass.

In some embodiments related to the methods for synthesizingoligonucleotides on a substrate having a functionalized surface asdescribed herein, the volume of the drop of the first and/or the secondreagents is at least 2 pl. In some embodiments, the volume of the dropis about 40 pl. In some embodiments, the volume of the drop is at most100 pl. In some embodiments, the microchannels comprise a density of thenominal arclength of the perimeter of at least 0.01 μm/μm². In someembodiments, the microchannels comprise a density of the nominalarclength of the perimeter of at least 0.001 μm/μm². In someembodiments, the functionalized surface comprises a nominal surface areaof at least 1 μm² per 1.0 μm² of planar surface area of the substrate.In some embodiments, the functionalized surface comprises a nominalsurface area of at least 1.25 μm² per 1.0 μm² of planar surface area ofthe substrate. In some embodiments, the functionalized surface comprisesa nominal surface area of at least 1.45 μm² per 1.0 μm² of planarsurface area of the substrate. In some embodiments, the pressuresurrounding the substrate is reduced to less than 1 mTorr. It is notedthat any of the embodiments described herein can be combined with any ofthe methods, devices, arrays, substrates or systems provided in thecurrent invention.

In some embodiments, the method for synthesizing oligonucleotides on asubstrate having a functionalized surface as described herein furthercomprises coupling at least a first building block originating from thefirst drop to a growing oligonucleotide chain on the first locus. Insome embodiments, the building blocks comprise a blocking group. In someembodiments, the blocking group comprises an acid-labile DMT. In someembodiments, the acid-labile DMT comprises 4,4′-dimethoxytrityl. In someembodiments, the method for synthesizing oligonucleotides on a substratehaving a functionalized surface as described herein further comprisesoxidation or sulfurization. In some embodiments, the method forsynthesizing oligonucleotides on a substrate having a functionalizedsurface as described herein further comprises chemically cappinguncoupled oligonucleotide chains. In some embodiments, the method forsynthesizing oligonucleotides on a substrate having a functionalizedsurface as described herein further comprises removing the blockinggroup, thereby deblocking the growing oligonucleotide chain. In someembodiments, the position of the substrate during the negative pressureapplication is within 10 cm of the position of the substrate during thecoupling step. In some embodiments, the position of the substrate duringthe negative pressure application is within 10 cm of the position of thesubstrate during the oxidation step. In some embodiments, the positionof the substrate during the negative pressure application is within 10cm of the position of the substrate during the capping step. In someembodiments, the position of the substrate during the negative pressureapplication is within 10 cm of the position of the substrate during thedeblocking step. In some embodiments, the first locus resides on amicrostructure fabricated into the substrate surface. In someembodiments, at least one reagent for the oxidation step is provided byflooding the microstructure with a solution comprising the at least onereagent. In some embodiments, at least one reagent for the capping stepis provided by flooding the microstructure with a solution comprisingthe at least one reagent. In some embodiments, the first locus resideson a microstructure fabricated into the substrate surface and at leastone reagent for the deblocking step can be provided by flooding themicrostructure with a solution comprising the at least one reagent. Insome embodiments, the method for synthesizing oligonucleotides on asubstrate having a functionalized surface as described herein furthercomprises enclosing the substrate within a sealed chamber. In someembodiments, the sealed chamber allows for purging of liquids from thefirst locus. In some embodiments, the method for synthesizingoligonucleotides on a substrate having a functionalized surface asdescribed herein further comprises draining a liquid through a drainthat is operably linked to the first locus. In some embodiments, afterapplying the negative pressure to the substrate, the moisture content onthe substrate is less than 1 ppm. In some embodiments, the surfaceenergy is increased corresponding to a water contact angle of less than20 degree. It is noted that any of the embodiments described herein canbe combined with any of the methods, devices, arrays, substrates orsystems provided in the current invention.

In yet another aspect, the present invention provides a method ofdepositing reagents to a plurality of resolved loci. The methodcomprises applying through an inkjet pump at least one drop of a firstreagent to a first locus of the plurality of loci; applying through aninkjet pump at least one drop of a second reagent to a second locus ofthe plurality of resolved loci. In some embodiments, the second locus isadjacent to the first locus. In still some embodiments, the first andsecond reagents are different. In still yet some embodiments, the firstand second loci reside on microstructures fabricated into a supportsurface. In yet some embodiments, the microstructures comprise at leastone channel that is more than 100 μm deep.

In practicing any of the methods of depositing reagents to a pluralityof resolved loci as described herein, in some embodiments, themicrostructures comprise at least two channels in fluidic communicationwith each other. In some embodiments, the at least two channels comprisetwo channels with different width. In some embodiments, the at least twochannels comprise two channels with different length. In someembodiments, the first locus receives less than 0.1% of the secondreagent and the second locus receives less than 0.1% of the firstreagent. In some embodiments, the loci comprise a density of the nominalarclength of the perimeter of at least 0.01 μm/square μm. In someembodiments, the loci comprise a density of the nominal arclength of theperimeter of at least 0.001 μm/square μm. In some embodiments, the firstand second loci comprise a coating of reagents. In some embodiments, thecoating of reagents is covalently linked to the substrate. In someembodiments, the coating of reagents comprises oligonucleotides. In someembodiments, at least one of the channels is longer than 100 μm. In someembodiments, at least one of the channels is shorter than 1000 μm. Insome embodiments, at least one of the channels is wider than 50 μm indiameter. In some embodiments, at least one of the channels is narrowerthan 100 μm in diameter. In some embodiments, the support surfacecomprises a material selected from the group consisting of silicon,polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides,PDMS, and glass. In some embodiments, the plurality of resolved loci isat a density of at least 1/mm². In some embodiments, the plurality ofresolved loci is at a density of at least 100/mm². In some embodiments,the volume of the drop is at least 2 pl. In some embodiments, the volumeof the drop is about 40 pl. In some embodiments, the volume of the dropis at most 100 pl. It is noted that any of the embodiments describedherein can be combined with any of the methods, devices, arrays,substrates or systems provided in the current invention.

In still yet another aspect, the present invention provides amicrofluidic system. The microfluidic system comprises a first surfacewith a plurality of microwells at a density of at least 10 per mm²; anda droplet inside one of the plurality of microwells. In someembodiments, the droplet inside one of the plurality of microwells has aReynolds number at a range of about 1-1000. In some embodiments, theplurality of microwells is at a density of at least 1 per mm². In someembodiments, plurality of microwells is at a density of at least 10 permm².

In some embodiments related to the microfluidic system as providedherein, the microfluidic system further comprises an inkjet pump. Insome embodiments, the droplet is deposited by the inkjet pump. In someembodiments, the droplet is moving in the lower half of a firstmicrowell dimension. In some embodiments, the droplet is moving in themiddle third of a first microwell dimension. In some embodiments, theplurality of microwells is at a density of at least 100 per mm². In someembodiments, the first microwell dimension is larger than the droplet.In some embodiments, the microwell is longer than 100 μm. In someembodiments, the microwell is shorter than 1000 μm. In some embodiments,the microwell is wider than 50 μm in diameter. In some embodiments, themicrowell is narrower than 100 μm in diameter. In some embodiments, thevolume of the droplet is at least 2 pl. In some embodiments, the volumeof the droplet is about 40 pl. In some embodiments, the volume of thedroplet is at most 100 pl. In some embodiments, each of the plurality ofmicrowells is fluidically connected to at least one microchannel. Insome embodiments, the at least one microchannel is coated with a moietythat increases surface energy. In some embodiments, the moiety is achemically inert moiety. In some embodiments, the surface energycorresponds to a water contact angle of less than 20 degree. In someembodiments, the microwells are formed on a solid support comprising amaterial selected from the group consisting of silicon, polystyrene,agarose, dextran, cellulosic polymers, polyacrylamides, PDMS, and glass.In some embodiments, the microchannels comprise a density of the nominalarclength of the perimeter of at least 0.01 μm/square μm. In someembodiments, the microchannels comprise a density of the nominalarclength of the perimeter of 0.001 μm/μm². In some embodiments, thesurface coated with the moiety comprises a nominal surface area of atleast 1 μm² per 1.0 μm² of planar surface area of the first surface. Insome embodiments, the surface coated with the moiety comprises a nominalsurface area of at least 1.25 μm² per 1.0 μm² of planar surface area ofthe first surface. In some embodiments, the surface coated with themoiety comprises a nominal surface area of at least 1.45 μm² per 1.0 μm²of planar surface area of the first surface. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays, substrates or systems provided in the currentinvention. In some embodiments, the droplet comprises a reagent thatenables oligonucleotide synthesis. In some embodiments, the reagent is anucleotide or nucleotide analog.

In another aspect, the present invention provides a method of depositingdroplets to a plurality of microwells. The method comprises applyingthrough an inkjet pump at least one droplet to a first microwell of theplurality of microwells. In some cases, the droplet inside one of theplurality of microwells has a Reynolds number at a range of about1-1000. In some embodiments, the plurality of microwells has a densityof at least 1/mm². In yet some cases, the plurality of microwells has adensity of at least 10/mm².

In practicing any of the methods of depositing droplets to a pluralityof microwells as provided herein, the plurality of microwells can have adensity of at least 100/mm². In some embodiments, the microwell islonger than 100 μm. In some embodiments, the microwell is shorter than1000 μm. In some embodiments, the microwell is wider than 50 μm indiameter. In some embodiments, the microwell is narrower than 100 μm indiameter. In some embodiments, the droplet is applied at a velocity ofat least 2 m/sec. In some embodiments, the volume of the droplet is atleast 2 pl. In some embodiments, the volume of the droplet is about 40pl. In some embodiments, the volume of the droplet is at most 100 pl. Insome embodiments, each of the plurality of microwells is fluidicallyconnected to at least one microchannel. In some embodiments, the atleast one microwell is coated with a moiety that increases surfaceenergy. In some embodiments, the moiety is a chemically inert moiety. Insome embodiments, the surface energy corresponds to a water contactangle of less than 20 degree. In some embodiments, the microwells areformed on a solid support comprising a material selected from the groupconsisting of silicon, polystyrene, agarose, dextran, cellulosicpolymers, polyacrylamides, PDMS, and glass. In some embodiments, themicrochannels comprise a density of the nominal arclength of theperimeter of at least 0.01 μm/square μm. In some embodiments, themicrochannels comprise a density of the nominal arclength of theperimeter of at least 0.001 μm²m/m². In some embodiments, the surfacecoated with the moiety comprises a nominal surface area of at least 1μm² per 1.0 μm² of planar surface area of the first surface. In someembodiments, the surface coated with the moiety comprises a nominalsurface area of at least 1.25 μm² per 1.0 μm² of planar surface area ofthe first surface. In some embodiments, the surface coated with themoiety comprises a nominal surface area of at least 1.45 μm² per 1.0 μm²of planar surface area of the first surface. In some embodiments, adroplet inside a microwell is traveling in the middle third of themicrowell. In some embodiments, a droplet inside a microwell istraveling in the bottom half of the microwell. In some embodiments,droplet comprises a reagent that enables oligonucleotide synthesis. Insome embodiments, the reagent is a nucleotide or nucleotide analog. Itis noted that any of the embodiments described herein can be combinedwith any of the methods, devices, arrays, substrates or systems providedin the current invention.

In another aspect, the present invention also provides a method ofpartitioning. The method of partitioning comprises contacting a firstsurface comprising a liquid at a first plurality of resolved loci with asecond surface comprising a second plurality of resolved loci;determining a velocity of release such that a desired fraction of theliquid can be transferred from the first plurality of resolved loci tothe second plurality of resolved loci; and detaching the second surfacefrom the first surface at said velocity. In some embodiments, the firstsurface comprises a first surface tension with the liquid, and thesecond surface can comprise a second surface tension with the liquid.

In practicing any of the methods of partitioning as provided herein, aportion of the first surface can be coated with a moiety that increasessurface tension. In some embodiments, the moiety is a chemically inertmoiety. In some embodiments, the surface tension of the first surfacecorresponds to a water contact angle of less than 20 degree. In someembodiments, the surface tension of the second surface corresponds to awater contact angle of more than 90 degree. In some embodiments, thefirst surface comprises a material selected from the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, PDMS, and glass. In some embodiments, the plurality ofresolved loci comprises a density of the nominal arclength of theperimeter of at least 0.01 μm/μm². In some embodiments, the plurality ofresolved loci comprises a density of the nominal arclength of theperimeter of at least 0.001 μm/μm². In some embodiments, the surfacecoated with the moiety comprises a nominal surface area of at least 1μm² per 1.0 μm² of planar surface area of the first surface. In someembodiments, the surface coated with the moiety comprises a nominalsurface area of at least 1.25 μm² per 1.0 μm² of planar surface area ofthe first surface. In some embodiments, the surface coated with themoiety comprises a nominal surface area of at least 1.45 μm² per 1.0 μm²of planar surface area of the first surface. In some embodiments, thefirst plurality of resolved loci is at a density of at least 1/mm². Insome embodiments, the first plurality of resolved loci is at a densityof at least 100/mm². In some embodiments, the first or the secondsurface comprises microchannels holding at least a portion of theliquid. In some embodiments, the first or the second surface comprisesnanoreactors holding at least a portion of the liquid. In someembodiments, the method of partitioning as described herein furthercomprises contacting a third surface with a third plurality of resolvedloci. In some embodiments, the liquid comprises a nucleic acid. In someembodiments, the desired fraction is more than 30%. In some embodiments,the desired fraction is more than 90%. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays, substrates or systems provided in the currentinvention.

In yet another aspect, the present invention also provides a method ofmixing as described herein. The method comprises: (a) providing a firstsubstrate comprising a plurality of microstructures fabricated thereto;(b) providing a second substrate comprising a plurality of resolvedreactor caps; (c) aligning the first and second substrates such that afirst reactor cap of the plurality can be configured to receive liquidfrom n microstructures in the first substrate; and (d) delivering liquidfrom the n microstructures into the first reactor cap, thereby mixingliquid from the n microstructures forming a mixture.

In practicing any of the methods of mixing as described herein, theplurality of resolved reactor caps can be at a density of at least0.1/mm². In some embodiments, the plurality of resolved reactor caps areat a density of at least 1/mm². In some embodiments, plurality ofresolved reactor caps are at a density of at least 10/mm². In someembodiments, each of the plurality of microstructures can comprise atleast two channels of different width. In some embodiments, the at leastone of the channels is longer than 100 μm. In some embodiments, the atleast one of the channels is shorter than 1000 μm. In some embodiments,the at least one of the channels is wider than 50 μm in diameter. Insome embodiments, the at least one of the channels is narrower than 100μm in diameter. In some embodiments, the at least one of the channels iscoated with a moiety that increases surface energy. In some embodiments,the moiety is a chemically inert moiety. In some embodiments, themicrostructures are formed on a solid support comprising a materialselected from the group consisting of silicon, polystyrene, agarose,dextran, cellulosic polymers, polyacrylamides, PDMS, and glass. In someembodiments, the microchannels comprise a density of the nominalarclength of the perimeter of at least 0.01 μm/square μm. In someembodiments, the microchannels comprise a density of the nominalarclength of the perimeter of at least 0.001 μm/m². In some embodiments,the surface coated with the moiety comprises a nominal surface area ofat least 1 μm² per 1.0 μm² of planar surface area of the first surface.In some embodiments, the surface coated with the moiety comprises anominal surface area of at least 1.25 μm² per 1.0 μm² of planar surfacearea of the first surface. In some embodiments, the surface coated withthe moiety comprises a nominal surface area of at least 1.45 μm² per 1.0μm² of planar surface area of the first surface. In some embodiments,the plurality of microstructures comprises a coating of reagents. Insome embodiments, the coating of reagents is covalently linked to thefirst surface. In some embodiments, the coating of reagents comprisesoligonucleotides. In some embodiments, the microstructures are at adensity of at least 1/mm². In some embodiments, the microstructures areat a density of at least 100/mm².

In some embodiments related to the methods of mixing as describedherein, after step (c), which is aligning the first and secondsubstrates such that a first reactor cap of the plurality can beconfigured to receive liquid from n microstructures in the firstsubstrate, there is a gap of less than 100 μm between the first and thesecond substrates. In some embodiments, after step (c), there is a gapof less than 50 μm between the first and the second substrates. In someembodiments, after step (c), there is a gap of less than 20 μm betweenthe first and the second substrates. In some embodiments, after step(c), there is a gap of less than 10 μm between the first and the secondsubstrates. In some embodiments, the mixture partially spreads into thegap. In some embodiments, the method of mixing further comprises sealingthe gap by bringing the first and the second substrate closer together.In some embodiments, one of the two channels is coated with a moietythat increases surface energy corresponding to a water contact angle ofless than 20 degree. In some embodiments, the moiety is a chemicallyinert moiety. In some embodiments, the delivering is performed bypressure. In some embodiments, the volume of the mixture is greater thanthe volume of the reactor cap. In some embodiments, the liquid comprisesa nucleic acid. In some embodiments, n is at least 10. In someembodiments, n is at least 25. In some embodiments, n, the number ofmicrostructures from which the liquid is mixed forming a mixture, can beat least 50. In some embodiments, n is at least 75. In some embodiments,n is at least 100. It is noted that any of the embodiments describedherein can be combined with any of the methods, devices, arrays,substrates or systems provided in the current invention.

In yet another aspect, the present invention also provides a method ofsynthesizing n-mer oligonucleotides on a substrate as described herein.The method comprises: providing a substrate with resolved loci that arefunctionalized with a chemical moiety suitable for nucleotide coupling;and coupling at least two building blocks to a plurality of growingoligonucleotide chains each residing on one of the resolved lociaccording to a locus specific predetermined sequence withouttransporting the substrate between the couplings of the at least twobuilding blocks, thereby synthesizing a plurality of oligonucleotidesthat are n basepairs long.

In practicing any of the methods of synthesizing n-mer oligonucleotideson a substrate as described herein, the method can further comprisecoupling at least two building blocks to a plurality of growingoligonucleotide chains each residing on one of the resolved loci at arate of at least 12 nucleotides per hour. In some embodiments, themethod further comprises coupling at least two building blocks to aplurality of growing oligonucleotide chains each residing on one of theresolved loci at a rate of at least 15 nucleotides per hour. In someembodiments, the method further comprises coupling at least two buildingblocks to a plurality of growing oligonucleotide chains each residing onone of the resolved loci at a rate of at least 20 nucleotides per hour.In some embodiments, the method further comprises coupling at least twobuilding blocks to a plurality of growing oligonucleotide chains eachresiding on one of the resolved loci at a rate of at least 25nucleotides per hour. In some embodiments, at least one resolved locuscomprises n-mer oligonucleotides deviating from the locus specificpredetermined sequence with an error rate of less than 1/500 bp. In someembodiments, at least one resolved locus comprises n-meroligonucleotides deviating from the locus specific predeterminedsequence with an error rate of less than 1/1000 bp. In some embodiments,at least one resolved locus comprises n-mer oligonucleotides deviatingfrom the locus specific predetermined sequence with an error rate ofless than 1/2000 bp. In some embodiments, the plurality ofoligonucleotides on the substrate deviate from respective locus specificpredetermined sequences at an error rate of less than 1/500 bp. In someembodiments, the plurality of oligonucleotides on the substrate deviatefrom respective locus specific predetermined sequences at an error rateof less than 1/1000 bp. In some embodiments, the plurality ofoligonucleotides on the substrate deviate from respective locus specificpredetermined sequences at an error rate of less than 1/2000 bp.

In some embodiments related to the method of synthesizing n-meroligonucleotides on a substrate as described herein, the building blockscomprise an adenine, guanine, thymine, cytosine, or uridine group, or amodified nucleotide. In some embodiments, the building blocks comprise amodified nucleotide. In some embodiments, the building blocks comprisedinucleotides. In some embodiments, the building blocks comprisephosphoramidite. In some embodiments, n is at least 100. In someembodiments, wherein n is at least 200. In some embodiments, n is atleast 300. In some embodiments, n is at least 400. In some embodiments,the substrate comprises at least 100,000 resolved loci and at least twoof the plurality of growing oligonucleotides are different from eachother. In some embodiments, the method further comprise vacuum dryingthe substrate before coupling. In some embodiments, the building blockscomprise a blocking group. In some embodiments, the blocking groupcomprises an acid-labile DMT. In some embodiments, the acid-labile DMTcomprises 4,4′-dimethoxytrityl. In some embodiments, the method furthercomprise oxidation or sulfurization. In some embodiments, the methodfurther comprise chemically capping uncoupled oligonucleotide chains. Insome embodiments, the method further comprise removing the blockinggroup, thereby deblocking the growing oligonucleotide chain. In someembodiments, the substrate comprises at least 10,000 vias providingfluid communication between a first surface of the substrate and asecond surface of the substrate. In some embodiments, the substratecomprises at least 100,000 vias providing fluid communication between afirst surface of the substrate and a second surface of the substrate. Insome embodiments, the substrate comprises at least 1,000,000 viasproviding fluid communication between a first surface of the substrateand a second surface of the substrate. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays, substrates or systems provided in the currentinvention.

In yet another aspect, the present invention also provides a method ofconstructing a gene library as described herein. The method comprises:entering at a first timepoint, in a computer readable non-transientmedium a list of genes, wherein the list comprises at least 100 genesand wherein the genes are at least 500 bp; synthesizing more than 90% ofthe list of genes, thereby constructing a gene library with deliverablegenes; preparing a sequencing library that represents the gene library;obtaining sequence information; selecting at least a subset of thedeliverable genes based on the sequence information; and delivering theselected deliverable genes at a second timepoint, wherein the secondtimepoint is less than a month apart from the first timepoint.

In practicing any of the methods of constructing a gene library asdescribed herein, the sequence information can be obtained vianext-generation sequencing. The sequence information can be obtained bySanger sequencing. In some embodiments, the method further comprisesdelivering at least one gene at a second timepoint. In some embodiments,at least one of the genes differ from any other gene by at least 0.1% inthe gene library. In some embodiments, each of the genes differ from anyother gene by at least 0.1% in the gene library. In some embodiments, atleast one of the genes differ from any other gene by at least 10% in thegene library. In some embodiments, each of the genes differ from anyother gene by at least 10% in the gene library. In some embodiments, atleast one of the genes differ from any other gene by at least 2 basepairs in the gene library. In some embodiments, each of the genes differfrom any other gene by at least 2 base pairs in the gene library. Insome embodiments, at least 90% of the deliverable genes are error free.In some embodiments, the deliverable genes comprise an error rate ofless than 1/3000 resulting in the generation of a sequence that deviatesfrom the sequence of a gene in the list of genes. In some embodiments,at least 90% of the deliverable genes comprise an error rate of lessthan 1 in 3000 bp resulting in the generation of a sequence thatdeviates from the sequence of a gene in the list of genes. In someembodiments, a subset of the deliverable genes are covalently linkedtogether. In some embodiments, a first subset of the list of genesencode for components of a first metabolic pathway with one or moremetabolic end products. In some embodiments, the method furthercomprises selecting of the one or more metabolic end products, therebyconstructing the list of genes. In some embodiments, the one or moremetabolic end products comprise a biofuel. In some embodiments, a secondsubset of the list of genes encode for components of a second metabolicpathway with one or more metabolic end products. In some embodiments,the list comprises at least 500, genes. In some embodiments, the listcomprises at least 5000 genes. In some embodiments, the list comprisesat least 10000 genes. In some embodiments, the genes are at least 1 kb.In some embodiments, the genes are at least 2 kb. In some embodiments,the genes are at least 3 kb. In some embodiments, the second timepointis less than 25 days apart from the first timepoint. In someembodiments, the second timepoint is less than 5 days apart from thefirst timepoint. In some embodiments, the second timepoint is less than2 days apart from the first timepoint. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays, substrates or systems provided in the currentinvention.

Provided herein, in some embodiments, is a microfluidic device fornucleic acid synthesis, comprising a substantially planar substrateportion comprising n groupings of m microfluidic connections betweenopposite surfaces, wherein each one of the n*m microfluidic connectionscomprises a first channel and a second channel, and wherein the firstchannel within each of the n groupings is common to all m microfluidicconnections, wherein the plurality of microfluidic connections span thesubstantially planar substrate portion along the smallest dimension ofthe substrate, and wherein n and m are at least 2. In some embodiments,the second channel is functionalized with a coating that is capable offacilitating the attachment of an oligonucleotide to the device. In someembodiments, the device further comprises a first oligonucleotide thatis attached to the second channels in k of the n groupings. In someembodiments, k is 1. In some embodiments, the device further comprises asecond oligonucleotide that is attached to 1 of the n groupings. In someembodiments, 1 is 1. In some embodiments, the none of the groupings inthe 1 groupings are in the k groupings.

In some embodiments, the oligonucleotide is at least 10 nucleotides, 25nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 125nucleotides, 150 nucleotides, or 200 nucleotides long.

In some embodiments, the first and the second oligonucleotides differ byat least 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or10 nucleotides.

In some embodiments, the n*m microfluidic connections are at most 5 mm,1.5 mm, 1.0 mm, or 0.5 mm long. In some embodiments, the first channelwithin each of the n groupings is at most 5 mm, 1.5 mm, 1.0 mm, or 0.5mm long. In some embodiments, the first channel within each of the ngroupings is at least 0.05 mm, 0.75 mm, 0.1 mm, 0.2 mm, 0.3 mm, or 0.4mm long. In some embodiments, the second channel in each of the n*mmicrofluidic connections is at most 0.2 mm, 0.1 mm, 0.05 mm, 0.04 mm, or0.03 mm long. In some embodiments, the second channel in each of the n*mmicrofluidic connections is at least 0.001 mm, 0.005 mm, 0.01 mm, 0.02mm, or 0.03 mm long. In some embodiments, the cross section of the firstchannel within each of the n groupings is at least 0.01 mm, 0.025 mm,0.05 mm, or 0.075 mm. In some embodiments, the cross section of thefirst channel within each of the n groupings is at most 1 mm, 0.5 mm,0.25 mm, 0.1 mm, or 0.075 mm. In some embodiments, the cross section ofthe second channel in each of the n*m microfluidic connections is atleast 0.001 mm, 0.05 mm, 0.01 mm, 0.015 mm, or 0.02 mm. In someembodiments, the cross section of the second channel in each of the n*mmicrofluidic connections is at most 0.25 mm, 0.125 mm, 0.050 mm, 0.025mm, 0.02 mm. In some embodiments, the standard deviation in the crosssection of the second channels in each of the n*m microfluidicconnections is less than 25%, 20%, 15%, 10%, 5%, or 1% of the mean ofthe cross section. In some embodiments, the variation in the crosssection within at least 90% of the second channels of the n*mmicrofluidic connections is at most 25%, 20%, 15%, 10%, 5%, or 1%.

In some embodiments, n is at least 10, 25, 50, 100, 1000, or 10000. Insome embodiments, m is at least 3, 4, or 5.

In some embodiments, the substrate comprises at least 5%, 10%, 25%, 50%,80%, 90%, 95%, or 99% silicon.

In some embodiments, at least 90% of the second channels of the n*mmicrofluidic connections is functionalized with a moiety that increasessurface energy. In some embodiments, the surface energy is increased toa level corresponding to a water contact angle of less than 75, 50, 30,or 20 degrees.

In some embodiments, the aspect ratio for at least 90% of the secondchannels of the n*m microfluidic connections is less than 1, 0.5, or0.3. In some embodiments, the aspect ratio for at least 90% of the firstchannels in the n groupings is less than 0.5, 0.3, or 0.2.

In some embodiments, the total length of at least 10%, 25%, 50%, 75%,90%, or 95% of the n*m fluidic connections are within 10%, 20%, 30%,40%, 50%, 100%, 200%, 500%, or 1000% of the smallest dimension of thesubstantially planar substrate.

In some embodiments, the substantially planar portion of the device isfabricated from a SOI wafer.

In another aspect, the invention relates to a method of nucleic acidamplification, comprising: (a) providing a sample comprising ncircularized single stranded nucleic acids, each comprising a differenttarget sequence; (b) providing a first adaptor that is hybridizable toat least one adaptor hybridization sequence on m of the n circularizedsingle stranded nucleic acids; (c) providing conditions suitable forextending the first adaptor using the m circularized single strandednucleic acids as a template, thereby generating m single strandedamplicon nucleic acids, wherein each of the m single stranded ampliconnucleic acids comprises a plurality of replicas of the target sequencefrom its template; (d) providing a first auxiliary oligonucleotide thatis hybridizable to the first adaptor; and (e) providing a first agentunder conditions suitable for the first agent to cut the m singlestranded amplicon nucleic acids at a plurality of cutting sites, therebygenerating a plurality of single stranded replicas of the targetsequences in the m circularized single stranded nucleic acids. In someembodiments, n or m is at least 2. In some embodiments, n or m is atleast 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300,400, or 500. In some embodiments, m is less than n. In some embodiments,the sample comprising the n circularized single stranded nucleic acid isformed by providing at least n linear single stranded nucleic acids,each comprising one of the different target sequences and circularizingthe n linear single stranded nucleic acids, thereby generating the ncircularized single stranded nucleic acids. In some embodiments, thefirst adaptor is hybridizable to both ends of the n linear singlestranded nucleic acids concurrently. In some embodiments, the differenttarget sequences in the n linear single stranded nucleic acids areflanked by a first and a second adaptor hybridization sequence. In someembodiments, the at least n linear single stranded nucleic acids aregenerated by de novo oligonucleotide synthesis. In some embodiments, thefirst adaptor hybridization sequence in each of the n linear singlestranded nucleic acids differ by no more than two nucleotide bases. Insome embodiments, the first or the second adaptor hybridization sequenceis at least 5 nucleotides long. In some embodiments, the first or thesecond adaptor hybridization sequence is at most 75, 50, 45, 40, 35, 30,or 25 nucleotides long. In some embodiments, the ends of the n linearsingle stranded nucleic acids pair with adjacent bases on the firstadaptor when the first adaptor is hybridized to the both ends of thelinear single stranded nucleic acid concurrently. In some embodiments,the locations of the plurality of cutting sites are such that theadaptor hybridization sequence is severed from at least 5% of aremainder sequence portion of the m circularized single stranded nucleicacid replicas. In some embodiments, at least 5% of the sequence of the mcircularized single stranded nucleic acid replicas other than the atleast one adaptor hybridization sequence remains uncut. In someembodiments, the locations of the plurality of cutting sites are outsidethe at least one adaptor hybridization sequence. In some embodiments,the locations of the plurality of cutting sites are independent of thetarget sequences. In some embodiments, the locations of the plurality ofcutting sites are determined by at least one sequence element within thesequence of the first adaptor or the first auxiliary oligonucleotide. Insome embodiments, the sequence element comprises a recognition site fora restriction endonuclease. In some embodiments, the first auxiliaryoligonucleotide or the first adaptor oligonucleotide comprises arecognition site for a Type IIS restriction endonuclease. In someembodiments, the recognition sites are at least 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotides away from the cutting sites. In some embodiments,the plurality of cutting sites are at junctures of single and doublestranded nucleic acids. In some embodiments, the double stranded nucleicacids comprise the first adaptor and the first auxiliaryoligonucleotide. In some embodiments, the single stranded nucleic acidsconsists essentially of the m different target sequences. In someembodiments, the m different target sequences have at most 95% pairwisesimilarity. In some embodiments, the m different target sequences haveat most 90% pairwise similarity. In some embodiments, the m differenttarget sequences have at most 80% pairwise similarity. In someembodiments, the m different target sequences have at most 50% pairwisesimilarity. In some embodiments, generating the m single strandedamplicon nucleic acid comprises strand displacement amplification. Insome embodiments, the first auxiliary oligonucleotide comprises anaffinity tag. In some embodiments, the affinity tag comprises biotin orbiotin derivative. In some embodiments, the method further comprisesisolating double stranded nucleic acids from the sample. In someembodiments, the isolating comprises affinity purification,chromatography, or gel purification. In some embodiments, the firstagent comprises a restriction endonuclease. In some embodiments, thefirst agent comprises at least two restriction endonucleases. In someembodiments, the first agent comprises a Type IIS restrictionendonuclease. In some embodiments, the first agent comprises a nickingendonuclease. In some embodiments, the first agent comprises at leasttwo nicking endonucleases. In some embodiments, the first agentcomprises at least one enzyme selected from the group consisting ofMlyI, SchI, AlwI, BccI, BceAI, BsmAI, BsmFI, FokI, HgaI, PleI, SfaNI,BfuAI, BsaI, BspMI, BtgZI, EarI, BspQI, SapI, SgeI, BceFI, BslFI,BsoMAI, Bst71I, FaqI, AceIII, BbvII, BveI, LguI, BfuCI, DpnII, FatI,MboI, MluCI, Sau3AI, Tsp509I, BssKI, PspGI, StyD4I, Tsp45I, AoxI, BscFI,Bsp143I, BssMI, BseENII, BstMBI, Kzo9I, NedII, Sse9I, TasI, TspEI, AjnI,BstSCI, EcoRII, MaeIII, NmuCI, Psp6I, MnlI, BspCNI, BsrI, BtsCI, HphI,HpyAV, MboII, AcuI, BciVI, BmrI, BpmI, BpuEI, BseRI, BsgI, BsmI, BsrDI,BtsI, EciI, MmeI, NmeAIII, Hin4II, TscAI, Bce83I, BmuI, BsbI, BscCI,NlaIII, Hpy99I, TspRI, FaeI, Hin1II, Hsp92II, SetI, TaiI, TscI, TscAI,TseFI, Nb.BsrDI, Nb.BtsI, AspCNI, BscGI, BspNCI, EcoHI, FinI, TsuI,UbaF11I, UnbI, Vpak11AI, BspGI, DrdII, Pfl1108I, UbaPI, Nt.AlwI,Nt.BsmAI, Nt.BstNBI, and Nt.BspQI, and variants thereof. In someembodiments, the first agent comprises essentially the same function,recognizes the same or essentially the same recognition sequence, orcuts at the same or essentially same cutting site, as any of the listedfirst agents and variants. In some embodiments, the at least tworestriction enzymes comprise MlyI and BciVI or BfuCI and MlyI. In someembodiments, the method further comprises (a) partitioning the sampleinto a plurality of fractions; (b) providing at least one fraction witha second adaptor that is hybridizable to at least one adaptorhybridization sequence on k of the n different circularized singlestranded nucleic acids; (c) providing conditions suitable for extendingthe second adaptor using the k circularized single stranded nucleicacids as a template, thereby generating k single stranded ampliconnucleic acids, wherein the second single stranded amplicon nucleic acidcomprises a plurality of replicas of the target sequence from itstemplate; (d) providing a second auxiliary oligonucleotide that ishybridizable to the second adaptor; and (e) providing a second agentunder conditions suitable for the agent to cut the k single strandedamplicon nucleic acids at a second plurality of cutting sites, therebygenerating a plurality of single stranded replicas of the targetsequences in the k circularized single stranded nucleic acids. In someembodiments, the first and the second adaptors are the same. In someembodiments, the first and the second auxiliary oligonucleotides are thesame. In some embodiments, the first and the second agents are the same.In some embodiments, k+m is less than n. In some embodiments, k is atleast 2. In some embodiments, the sample comprising the n circularizedsingle stranded nucleic acid is formed by single stranded nucleic acidamplification. In some embodiments, the single stranded nucleic acidamplification comprises: (a) providing a sample comprising at least mcircularized single stranded precursor nucleic acids; (b) providing afirst precursor adaptor that is hybridizable to the m circularizedsingle stranded precursor nucleic acids; (c) providing conditionssuitable for extending the first precursor adaptor using the mcircularized single stranded precursor nucleic acids as a template,thereby generating m single stranded precursor amplicon nucleic acids,wherein the single stranded amplicon nucleic acid comprises a pluralityof replicas of the m circularized single stranded precursor nucleicacid; (d) providing a first precursor auxiliary oligonucleotide that ishybridizable to the first precursor adaptor; and (e) providing a firstprecursor agent under conditions suitable for the first precursor agentto cut the first single stranded precursor amplicon nucleic acid at aplurality of cutting sites, thereby generating the m linear precursornucleic acids. In some embodiments, the method further comprisescircularizing the m linear precursor nucleic acids, thereby formingreplicas of the m circularized single stranded precursor nucleic acids.In some embodiments, the m circularized single stranded precursornucleic acid is amplified by at least 10, 100, 250, 500, 750, 1000,1500, 2000, 3000, 4000, 5000, 10000-fold, or more in single strandedreplicas. In some embodiments, at least one of the m circularized singlestranded nucleic acids is at a concentration of about or at most about100 nM, 10 nM, 1 nM, 50 pM, 1 pM, 100 fM, 10 fM, 1 fM, or less. In someembodiments, circularizing comprises ligation. In some embodiments,ligation comprises the use of a ligase selected from the groupconsisting of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNAligase, Taq DNA ligase, and 9N DNA ligase.

In yet a further aspect, the invention, in various embodiments relatesto a kit comprising: (a) a first adaptor; (b) a first auxiliaryoligonucleotide that is hybridizable to the adaptor; (c) a ligase; and(d) a first cleaving agent, comprising at least one enzyme selected fromthe group consisting of MlyI, SchI, AlwI, BccI, BceAI, BsmAI, BsmFI,FokI, HgaI, PleI, SfaNI, BfuAI, BsaI, BspMI, BtgZI, EarI, BspQI, SapI,SgeI, BceFI, BslFI, BsoMAI, Bst71I, FaqI, AceIII, BbvII, BveI, LguI,BfuCI, DpnII, FatI, MboI, MluCI, Sau3AI, Tsp509I, BssKI, PspGI, StyD4I,Tsp45I, AoxI, BscFI, Bsp143I, BssMI, BseENII, BstMBI, Kzo9I, NedII,Sse9I, TasI, TspEI, AjnI, BstSCI, EcoRII, MaeIII, NmuCI, Psp6I, MnlI,BspCNI, BsrI, BtsCI, HphI, HpyAV, MboII, AcuI, BciVI, BmrI, BpmI, BpuEI,BseRI, BsgI, BsmI, BsrDI, BtsI, EciI, MmeI, NmeAIII, Hin4II, TscAI,Bce83I, BmuI, BsbI, BscCI, NlaIII, Hpy99I, TspRI, FaeI, Hin1II, Hsp92II,SetI, TaiI, TscI, TscAI, TseFI, Nb.BsrDI, Nb.BtsI, AspCNI, BscGI,BspNCI, EcoHI, FinI, TsuI, UbaF11I, UnbI, Vpak11AI, BspGI, DrdII,Pfl1108I, UbaPI, Nt.AlwI, Nt.BsmAI, Nt.BstNBI, and Nt.BspQI, andvariants thereof. In some embodiments, the first agent comprisesessentially the same function, recognizes the same or essentially thesame recognition sequence, or cuts at the same or essentially samecutting site as any of the listed first agents and variants. In someembodiments, the kit further comprises a second cleaving agent. In someembodiments, the second cleaving agent comprises and enzyme selectedfrom the group consisting of MlyI, SchI, AlwI, BccI, BceAI, BsmAI,BsmFI, FokI, HgaI, PleI, SfaNI, BfuAI, BsaI, BspMI, BtgZI, EarI, BspQI,SapI, SgeI, BceFI, BslFI, BsoMAI, Bst71I, FaqI, AceIII, BbvII, BveI,LguI, BfuCI, DpnII, FatI, MboI, MluCI, Sau3AI, Tsp509I, BssKI, PspGI,StyD4I, Tsp45I, AoxI, BscFI, Bsp143I, BssMI, BseENII, BstMBI, Kzo9I,NedII, Sse9I, TasI, TspEI, AjnI, BstSCI, EcoRII, MaeIII, NmuCI, Psp6I,MnlI, BspCNI, BsrI, BtsCI, HphI, HpyAV, MboII, AcuI, BciVI, BmrI, BpmI,BpuEI, BseRI, BsgI, BsmI, BsrDI, BtsI, EciI, MmeI, NmeAIII, Hin4II,TscAI, Bce83I, BmuI, BsbI, BscCI, NlaIII, Hpy99I, TspRI, FaeI, Hin1II,Hsp92II, SetI, TaiI, TscI, TscAI, TseFI, Nb.BsrDI, Nb.BtsI, AspCNI,BscGI, BspNCI, EcoHI, FinI, TsuI, UbaF11I, UnbI, Vpak11AI, BspGI, DrdII,Pfl1108I, UbaPI, Nt.AlwI, Nt.BsmAI, Nt.BstNBI, and Nt.BspQI, andvariants thereof. In some embodiments, the second agent comprisesessentially the same function, recognizes the same or essentially thesame recognition sequence, or cuts at the same or essentially samecutting site as any of the listed second agents and variants. In someembodiments, the first cleaving agents comprises MlyI. In someembodiments, the second cleaving agent comprises BciVI or BfuCI.

In yet another aspect, the invention relates to a method of nucleic acidamplification, comprising: (a) providing a sample comprising ncircularized single stranded nucleic acids, each comprising a differenttarget sequence; (b) providing a first adaptor that is hybridizable toat least one adaptor hybridization sequence on m of the n circularizedsingle stranded nucleic acids; (c) providing conditions suitable forextending the first adaptor using the m circularized single strandednucleic acids as a template, thereby generating m single strandedamplicon nucleic acids, wherein each of the m single stranded ampliconnucleic acids comprises a plurality of replicas of the target sequencefrom its template; (d) generating double stranded recognition sites fora first agent on the m single stranded amplicon nucleic acids; and (e)providing the first agent under conditions suitable for the first agentto cut the m single stranded amplicon nucleic acids at a plurality ofcutting sites, thereby generating a plurality of single strandedreplicas of the target sequences in the m circularized single strandednucleic acids. In some embodiments, the double stranded recognitionsites comprise a first portion of the first adaptor on a first strand ofthe double stranded recognition sites and a second strand of the firstadaptor on the second strand of the double stranded recognition sites.In some embodiments, the adaptor comprises a palindromic sequence. Insome embodiments, the double stranded recognition sites are generated byhybridizing the first and second portions of the first adaptor to eachother. In some embodiments, the m single stranded amplicon nucleic acidscomprise a plurality of double stranded self-hybridized regions.

In a yet further aspect, the invention relates to a method forgenerating a long nucleic acid molecule, the method comprising the stepsof: (a) providing a plurality of nucleic acids immobilized on a surface,wherein said plurality of nucleic acids comprises nucleic acids havingoverlapping complementary sequences; (b) releasing said plurality ofnucleic acids into solution; and (c) providing conditions promoting: i)hybridization of said overlapping complementary sequences to form aplurality of hybridized nucleic acids; and ii) extension or ligation ofsaid hybridized nucleic acids to synthesize the long nucleic acidmolecule.

In another aspect, the invention relates to an automated system capableof processing one or more substrates, comprising: an inkjet print headfor spraying a microdroplet comprising a chemical species on asubstrate; a scanning transport for scanning the substrate adjacent tothe print head to selectively deposit the microdroplet at specifiedsites; a flow cell for treating the substrate on which the microdropletis deposited by exposing the substrate to one or more selected fluids;an alignment unit for aligning the substrate correctly relative to theprint head each time when the substrate is positioned adjacent to theprint head for deposition; and not comprising a treating transport formoving the substrate between the print head and the flow cell fortreatment in the flow cell, wherein said treating transport and saidscanning transport are different elements.

In yet another aspect, the invention relates to an automated system forsynthesizing oligonucleotides on a substrate, said automated systemcapable of processing one or more substrates, comprising: an inkjetprint head for spraying a solution comprising a nucleoside or activatednucleoside on a substrate; a scanning transport for scanning thesubstrate adjacent to the print head to selectively deposit thenucleoside at specified sites; a flow cell for treating the substrate onwhich the monomer is deposited by exposing the substrate to one or moreselected fluids; an alignment unit for aligning the substrate correctlyrelative to the print head each time when the substrate is positionedadjacent to the print head for deposition; and not comprising a treatingtransport for moving the substrate between the print head and the flowcell for treatment in the flow cell, wherein said treating transport andsaid scanning transport are different elements.

In yet a further aspect, the invention relates to an automated systemcomprising: an inkjet print head for spraying a microdroplet comprisinga chemical species on a substrate; a scanning transport for scanning thesubstrate adjacent to the print head to selectively deposit themicrodroplet at specified sites; a flow cell for treating the substrateon which the microdroplet is deposited by exposing the substrate to oneor more selected fluids; and an alignment unit for aligning thesubstrate correctly relative to the print head each time when thesubstrate is positioned adjacent to the print head for deposition; andwherein the system does NOT comprise a treating transport for moving thesubstrate between the print head and the flow cell for treatment in theflow cell.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent or patent application with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A-1C demonstrates an example process outlining the gene synthesisand nanoreactor technologies. FIG. 1A illustrates an example process foroligonucleotide synthesis on a substrate using an inkjet printer; FIG.1B illustrates an example process for gene amplification in a resolvedenclosure, or a nanoreactor. FIG. 1C illustrates an example of the useof a plurality of wafers linking microfluidic reactions foroligonucleotide synthesis and gene assembly in parallel.

FIGS. 2A-2C are block diagrams demonstrating exemplary business processflows. Cloning of the synthesized genes may be skipped (FIG. 2B). InFIG. 2C, synthesized genes are cloned prior to shipment (FIG. 2C).

FIG. 3 demonstrates an exemplary outline of a system for oligonucleotidesynthesis, including a printer, e.g. inkjet printer, for reagentdeposition, a substrate (wafer), schematics outlining the alignment ofthe system elements in multiple directions, and exemplary setups forreagent flow.

FIG. 4 illustrates an example of the design microstructures built into asubstrate (oligonucleotide wafer reactor).

FIG. 5 is a diagram demonstrating an exemplary process for reagentdeposition into the microstructures illustrated in FIG. 4. The selectedarea for surface functionalization allows reagent spreading into thesmaller functionalized wells under wetting conditions.

FIG. 6A are illustrations further exemplifying the microstructuresillustrated in FIG. 4. FIG. 6B are illustrations of various alternativedesigns for the microstructures. FIG. 6C illustrates a layout design forthe microstructures on the substrate (wafer).

FIG. 7 illustrates an exemplary layout of reactor caps on a cappingelement.

FIG. 8 is a diagram demonstrating an exemplary process workflow for genesynthesis to shipment.

FIG. 9 part A show illustrations of an exemplary flowcell with lidopened or closed. FIG. 9 part B illustrates a cross-sectional view of anexemplary flowcell and waste collector assembly. FIG. 9 part Cillustrates a magnified cross-sectional view of an exemplary flowcelland waste collector assembly.

FIG. 10 part A illustrates an example of a single groove vacuum chuckwith a single 1-5 mm groove, 198 mm diameter. FIG. 10 part B illustratesa sintered metal insert in between a substrate (wafer) and the vacuumchuck and an optional thermal control element incorporated into thereceiving element. FIG. 10 part C illustrates a cross-sectional view ofthe single groove vacuum chuck exemplified in FIG. 10 part A.

FIG. 11 illustrates exemplary application standard phosphoramiditechemistry for oligonucleotide synthesis.

FIG. 12 illustrates an exemplary application of the polymerase chainassembly (PCA).

FIG. 13 are diagrams demonstrating the advantage of using longeroligonucleotides (e.g. about 300 bp) vs. shorter oligonucleotides (e.g.about 50 kb). Longer oligonucleotides can be used in the assembly ofgene products with reduced error.

FIG. 14 are diagrams demonstrating an exemplary combined application ofPCA and Gibson methods for assembly of oligonucleotides into geneproducts.

FIG. 15 is a diagram demonstrating an error correction method especiallysuited for application to gene synthesis products with higher errorrates.

FIG. 16 is a diagram demonstrating an error correction method especiallysuited for application to gene synthesis products with lower errorrates.

FIG. 17 is a diagram demonstrating the use of padlock probes for thegeneration of molecularly barcoded sequencing libraries and qualitycontrol (QC) processes comprising next generation sequencing (NGS).

FIG. 18 illustrates an example for an inkjet assembly, with 10 inkjetheads that have silicon orifice plates with 256 nozzles on 254 μmcenters, and 100 μm fly height.

FIG. 19 illustrates an example of a computer system that can be used inconnection with example embodiments of the present invention.

FIG. 20 is a block diagram illustrating a first example architecture ofa computer system 2000 that can be used in connection with exampleembodiments of the present invention.

FIG. 21 is a diagram demonstrating a network 2100 configured toincorporate a plurality of computer systems, a plurality of cell phonesand personal data assistants, and Network Attached Storage (NAS) thatcan be used in connection with example embodiments of the presentinvention.

FIG. 22 is a block diagram of a multiprocessor computer system 2200using a shared virtual address memory space that can be used inconnection with example embodiments of the present invention.

FIG. 23 is a diagram demonstrating exemplary steps constituting thefront end processing for the manufacturing of microstructures on asubstrate (e.g. silicon wafer).

FIG. 24 is a diagram demonstrating exemplary steps constituting the backend processing for the functionalizing of the microstructure surfaces ona substrate (e.g. silicon wafer).

FIGS. 25A-25C depict different views of a cluster comprising a highdensity of groupings. FIGS. 25D-25E depict different views of a diagramof a microfluidic device comprising a substantially planar substrateportion. FIG. 25F depicts the device view of a diagram of a microfluidicdevice comprising a substantially planar substrate portion having 108reaction wells and a designated area for a label. FIG. 25G depicts thedevice view of a cluster comprising 109 groupings.

FIG. 26A depicts a cross-section view of a diagram of a nanoreactor,where the view shows a row of the nanoreactor comprising 11 wells. FIG.26B depicts a device view of a diagram of a nanoreactor comprising 108raised wells. The detail F depicts a detailed view of one well of thenanoreactor. FIG. 26C depicts an angled device view of the nanoreactordiagram shown in FIG. 26B. FIG. 26D depicts a handle view of a diagramof a nanoreactor. The detail H depicts a detailed view of a fiducialmarking on the handle side of the nanoreactor. FIG. 26E depicts a deviceview of a diagram of nanoreactor comprising 108 wells and a label.

FIG. 27 illustrates in detail the design features of an exemplaryoligonucleotide synthesis device that is differentially functionalized.

FIG. 28 illustrates a workflow for the front-end manufacturing processfor the exemplary device in FIG. 15.

FIG. 29 illustrates an exemplary baseline process flow for the back-endmanufacturing of the exemplary oligonucleotide synthesis device of FIG.15 for differential functionalization.

FIG. 30 illustrates a functionalized surface with a controlled densityof active groups for nucleic acid synthesis.

FIG. 31 parts A-B shows an image of a device manufactured according tothe methods described herein.

FIG. 32 illustrates the design details of an exemplary nanoreactordevice.

FIG. 33 parts A-H illustrates an exemplary baseline process flow for thefront-end manufacturing of the exemplary device described in FIG. 20.

FIG. 34 parts A-D illustrates an exemplary baseline process flow for theback-end manufacturing of the exemplary nanoreactor device of FIG. 20for functionalization.

FIG. 35 illustrates the nanowells in a nanoreactor device manufacturedas described herein. FIG. 35 part B illustrates a close-up view of thenanowells illustrated in FIG. 35 part A.

FIG. 36 parts A-F illustrates various configurations for differentialfunctionalization. In each figure, the light shaded line indicates anactive surface, while a dark line indicates a passive surface.

FIG. 36 part A illustrates a uniformly functionalized surface. FIG. 36parts B-F illustrate differentially functionalized surfaces in variousconfigurations.

FIG. 37 parts A-F illustrate a process flow for device funtionalization.

FIG. 38 depicts an exemplary illustration of resist application, whereinresist is pulled into small structures and stopped by sharp edges.

FIG. 39 parts A-B illustrate use of underlying structures to either stopor wick the resist application in an exemplary embodiment.

FIG. 40 parts A-C illustrate post-lithographic resist patterns in anexemplary differential functionalization configuration. FIG. 40 part Aillustrates a bright field view of a post-lithographic resist pattern.FIG. 40 part B illustrates a dark field view of a post-lithographicresist pattern. FIG. 40 part C illustrates a cross-sectional schematicview of a post-lithographic resist pattern.

FIG. 41 parts A-C illustrate post-lithographic resist patterns inanother exemplary differential functionalization configuration. FIG. 41part A illustrates a bright field view of a post-lithographic resistpattern. FIG. 41 part B illustrates a dark field view of apost-lithographic resist pattern. FIG. 41 part C illustrates across-sectional schematic view of a post-lithographic resist pattern.

FIG. 42 parts A-C illustrate a post resist strip after functionalizationwith a fluorosilane. FIG. 42 part A illustrates a bright field view.FIG. 42 part B illustrates a dark field view. FIG. 42 part C illustratesa cross-sectional schematic view.

FIG. 43 parts A-C illustrate an exemplary oligonucleotide synthesisdevice (“Keratin chip”), fully loaded with DMSO. FIG. 43 part Aillustrates a bright field view of the Keratin chip fully loaded withDMSO. Hydrophilic and hydrophobic regions are indicated. FIG. 43 part Billustrates a dark field view of the Keratin chip fully loaded withDMSO. FIG. 43 part C illustrates a cross-sectional schematic view of theKeratin chip fully loaded with DMSO, indicating spontaneous wetting ofthe revolvers.

FIG. 44 parts A-F outline an exemplary process flow for configuration 6illustrated in FIG. 36.

FIG. 45 parts A-B indicate a spot sampling configuration from anoligonucleotide synthesis device (A) and corresponding BioAnalyzer data(B) for each of the five spots in FIG. 45 part A.

FIG. 46 indicates BioAnalyzer data of surface extracted 100-meroligonucleotides synthesized on a silicon oligonucleotide synthesisdevice.

FIG. 47 indicates BioAnalyzer data of surface extracted 100-meroligonucleotides synthesized on a silicon oligonucleotide synthesisdevice after PCR amplification.

FIG. 48 represents a sequence alignment for the samples taken from spot8, where “×” denotes a single base deletion, “star” denotes single basemutation, and “+” denotes low quality spots in Sanger sequencing.

FIG. 49 represents a sequence alignment for the samples taken from spot7, where “×” denotes a single base deletion, “star” denotes single basemutation, and “+” denotes low quality spots in Sanger sequencing.

FIG. 50 parts A-B provide BioAnalzyer results for a 100-meroligonucleotide synthesized on a three dimensional oligonucleotidedevice after extraction (part A) and after PCR amplification (part B).

FIG. 51 represents a sequence alignment map for a PCR amplified sampleof a 100-mer oligonucleotide that was synthesized on a 3Doligonucleotide device.

FIG. 52 represents correction results through the application of tworounds of error correction using CorrectASE.

FIG. 53 parts A-C illustrate a surface functionalization pattern in anexemplary differential functionalization configuration afterfunctionalization. FIG. 53 part A illustrates a bright field view. FIG.53 part B illustrates a dark field view. FIG. 53 part C illustrates across-sectional schematic view of the surface functionalization patternand an aqueous fluid bulging out avoiding hydrophobic regions.

FIG. 54 parts A-D depicts an exemplary workflow for functionalization ofan nanoreactor device. Cleaning is followed by resist deposition,functionalization, and finally a resist strip.

FIG. 55 depicts BioAnalyzer results for a number of oligonucleotidestransferred into individual nanoreactor wells from an oligonucleotidesynthesis device following a blotting method. FIG. 56 parts A-B depictalternate flow cell designs.

FIG. 56 part A depicts a line source/line drain design for a flowcell.

FIG. 56 part B depicts a point source/point drain design for a flowcell.

FIG. 57 illustrates an oligonucleotide synthesis device and ananoreactor device mounted in a configuration having a 50 um gap. In anexemplary embodiment, the devices are maintained in this configurationfor for 10 minutes.

FIG. 58 parts A-B show the redistribution of oligos over time, withoutbeing bound by theory, by diffusion, from an oligonucleotide synthesisdevice to a nanoreactor device.

FIG. 58 part A shows oligos concentrated in a liquid in the revolverchannels, and few or no oligonucleotides in a nanoreactor chamber. FIG.58 part B schematizes oligonucleotides uniformly distributed throughliquid in revolver chambers and in a nanoreactor chamber at a later timepoint relative to FIG. 58 part A.

FIG. 59 shows views of a nanoreactor well array used for gene assemblybefore and after a PCA reaction.

FIG. 60 parts A-C depict the results of the assembly of a gene invarious wells of a nanoreactor device. FIG. 60 part A depicts a devicein which oligos were synthesized. Wells 1-10 are marked. FIG. 60 part Bdepicts analysis of the genes assembled in the wells in FIG. 60 part A.Peaks corresponding to the gene in each well are labeled with the wellnumber. FIG. 60 part C depicts electrophoresis of the oligos analyzed inFIG. 60 part B.

FIG. 61 parts A-B present block views of a high capacity oligonucleotidesynthesis device consistent with the disclosure herein. FIG. 61 part Apresents a full, angled view of a block as disclosed herein. FIG. 61part B presents an angled view of a cross-sectional slice through ablock as disclosed herein.

FIG. 62 depicts a block view of another high capacity oligonucleotidesynthesis device consistent with the disclosure herein, having an arrayof posts on its surface, which increase surface area.

FIG. 63 depicts electrophoresis of amplified single stranded nucleicacids using rolling circle amplification, wherein the amplificationproduct is cut with various combinations of cleaving agents.

FIG. 64 parts A-F represent a method for the amplification of singlestranded nucleic acids.

FIG. 65 parts A-F represent method for the amplification of singlestranded nucleic acids, which may be coupled to the method illustratedin FIG. 64.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range to the tenth of the unitof the lower limit unless the context clearly dictates otherwise. Forexample, description of a range such as from 1 to 6 should be consideredto have specifically disclosed subranges such as from 1 to 3, from 1 to4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual values within that range, for example, 1.1, 2, 2.3, 5, and5.9. This applies regardless of the breadth of the range. The upper andlower limits of these intervening ranges may independently be includedin the smaller ranges, and are also encompassed within the invention,subject to any specifically excluded limit in the stated range. Wherethe stated range includes one or both of the limits, ranges excludingeither or both of those included limits are also included in theinvention, unless the context clearly dictates otherwise.

In one aspect, the present invention provides a gene library asdescribed herein. The gene library comprises a collection of genes. Insome embodiments, the collection comprises at least 100 differentpreselected synthetic genes that can be of at least 0.5 kb length withan error rate of less than 1 in 3000 bp compared to predeterminedsequences comprising the genes. In another aspect, the present inventionalso provides a gene library that comprises a collection of genes. Thecollection may comprise at least 100 different preselected syntheticgenes that can be each of at least 0.5 kb length. At least 90% of thepreselected synthetic genes may comprise an error rate of less than 1 in3000 bp compared to predetermined sequences comprising the genes.Desired predetermined sequences may be supplied by any method, typicallyby a user, e.g. a user entering data using a computerized system. Invarious embodiments, synthesized nucleic acids are compared againstthese predetermined sequences, in some cases by sequencing at least aportion of the synthesized nucleic acids, e.g. using next-generationsequencing methods. In some embodiments related to any of the genelibraries described herein, at least 90% of the preselected syntheticgenes comprise an error rate of less than 1 in 5000 bp compared topredetermined sequences comprising the genes. In some embodiments, atleast 0.05% of the preselected synthetic genes are error free. In someembodiments, at least 0.5% of the preselected synthetic genes are errorfree. In some embodiments, at least 90% of the preselected syntheticgenes comprise an error rate of less than 1 in 3000 bp compared topredetermined sequences comprising the genes. In some embodiments, atleast 90% of the preselected synthetic genes are error free orsubstantially error free. In some embodiments, the preselected syntheticgenes comprise a deletion rate of less than 1 in 3000 bp compared topredetermined sequences comprising the genes. In some embodiments, thepreselected synthetic genes comprise an insertion rate of less than 1 in3000 bp compared to predetermined sequences comprising the genes. Insome embodiments, the preselected synthetic genes comprise asubstitution rate of less than 1 in 3000 bp compared to predeterminedsequences comprising the genes. In some embodiments, the gene library asdescribed herein further comprises at least 10 copies of each syntheticgene. In some embodiments, the gene library as described herein furthercomprises at least 100 copies of each synthetic gene. In someembodiments, the gene library as described herein further comprises atleast 1000 copies of each synthetic gene. In some embodiments, the genelibrary as described herein further comprises at least 1000000 copies ofeach synthetic gene. In some embodiments, the collection of genes asdescribed herein comprises at least 500 genes. In some embodiments, thecollection comprises at least 5000 genes. In some embodiments, thecollection comprises at least 10000 genes. In some embodiments, thepreselected synthetic genes are at least 1 kb. In some embodiments, thepreselected synthetic genes are at least 2 kb. In some embodiments, thepreselected synthetic genes are at least 3 kb. In some embodiments, thepredetermined sequences comprise less than 20 bp in addition compared tothe preselected synthetic genes. In some embodiments, the predeterminedsequences comprise less than 15 bp in addition compared to thepreselected synthetic genes. In some embodiments, at least one of thesynthetic genes differs from any other synthetic gene by at least 0.1%.In some embodiments, each of the synthetic genes differs from any othersynthetic gene by at least 0.1%. In some embodiments, at least one ofthe synthetic genes differs from any other synthetic gene by at least10%. In some embodiments, each of the synthetic genes differs from anyother synthetic gene by at least 10%. In some embodiments, at least oneof the synthetic genes differs from any other synthetic gene by at least2 base pairs. In some embodiments, each of the synthetic genes differsfrom any other synthetic gene by at least 2 base pairs. In someembodiments, the gene library as described herein further comprisessynthetic genes that are of less than 2 kb with an error rate of lessthan 1 in 20000 bp compared to preselected sequences of the genes. Insome embodiments, a subset of the deliverable genes is covalently linkedtogether. In some embodiments, a first subset of the collection of genesencodes for components of a first metabolic pathway with one or moremetabolic end products. In some embodiments, the gene library asdescribed herein further comprises selecting of the one or moremetabolic end products, thereby constructing the collection of genes. Insome embodiments, the one or more metabolic end products comprise abiofuel. In some embodiments, a second subset of the collection of genesencodes for components of a second metabolic pathway with one or moremetabolic end products. In some embodiments, the gene library is in aspace that is less than 100 m³. In some embodiments, the gene library isin a space that is less than 1 m³. In some embodiments, the gene libraryis in a space that is less than 1 m³.

In another aspect, the present invention also provides a method ofconstructing a gene library. The method comprises the steps of: enteringbefore a first timepoint, in a computer readable non-transient medium atleast a first list of genes and a second list of genes, wherein thegenes are at least 500 bp and when compiled into a joint list, the jointlist comprises at least 100 genes; synthesizing more than 90% of thegenes in the joint list before a second timepoint, thereby constructinga gene library with deliverable genes. In some embodiments, the secondtimepoint is less than a month apart from the first timepoint.

In practicing any of the methods of constructing a gene library asprovided herein, the method as described herein further comprisesdelivering at least one gene at a second timepoint. In some embodiments,at least one of the genes differs from any other gene by at least 0.1%in the gene library. In some embodiments, each of the genes differs fromany other gene by at least 0.1% in the gene library. In someembodiments, at least one of the genes differs from any other gene by atleast 10% in the gene library. In some embodiments, each of the genesdiffers from any other gene by at least 10% in the gene library. In someembodiments, at least one of the genes differs from any other gene by atleast 2 base pairs in the gene library. In some embodiments, each of thegenes differs from any other gene by at least 2 base pairs in the genelibrary. In some embodiments, at least 90% of the deliverable genes areerror free. In some embodiments, the deliverable genes comprises anerror rate of less than 1/3000 resulting in the generation of a sequencethat deviates from the sequence of a gene in the joint list of genes. Insome embodiments, at least 90% of the deliverable genes comprise anerror rate of less than 1 in 3000 bp resulting in the generation of asequence that deviates from the sequence of a gene in the joint list ofgenes. In some embodiments, genes in a subset of the deliverable genesare covalently linked together. In some embodiments, a first subset ofthe joint list of genes encode for components of a first metabolicpathway with one or more metabolic end products. In some embodiments,any of the methods of constructing a gene library as described hereinfurther comprises selecting of the one or more metabolic end products,thereby constructing the first, the second or the joint list of genes.In some embodiments, the one or more metabolic end products comprise abiofuel. In some embodiments, a second subset of the joint list of genesencode for components of a second metabolic pathway with one or moremetabolic end products. In some embodiments, the joint list of genescomprises at least 500 genes. In some embodiments, the joint list ofgenes comprises at least 5000 genes. In some embodiments, the joint listof genes comprises at least 10000 genes. In some embodiments, the genescan be at least 1 kb. In some embodiments, the genes are at least 2 kb.In some embodiments, the genes are at least 3 kb. In some embodiments,the second timepoint is less than 25 days apart from the firsttimepoint. In some embodiments, the second timepoint is less than 5 daysapart from the first timepoint. In some embodiments, the secondtimepoint is less than 2 days apart from the first timepoint. It isnoted that any of the embodiments described herein can be combined withany of the methods, devices or systems provided in the currentinvention.

In another aspect, a method of constructing a gene library is providedherein. The method comprises the steps of: entering at a firsttimepoint, in a computer readable non-transient medium a list of genes;synthesizing more than 90% of the list of genes, thereby constructing agene library with deliverable genes; and delivering the deliverablegenes at a second timepoint. In some embodiments, the list comprises atleast 100 genes and the genes can be at least 500 bp. In still yet someembodiments, the second timepoint is less than a month apart from thefirst timepoint.

In practicing any of the methods of constructing a gene library asprovided herein, in some embodiments, the method as described hereinfurther comprises delivering at least one gene at a second timepoint. Insome embodiments, at least one of the genes differs from any other geneby at least 0.1% in the gene library. In some embodiments, each of thegenes differs from any other gene by at least 0.1% in the gene library.In some embodiments, at least one of the genes differs from any othergene by at least 10% in the gene library. In some embodiments, each ofthe genes differs from any other gene by at least 10% in the genelibrary. In some embodiments, at least one of the genes differs from anyother gene by at least 2 base pairs in the gene library. In someembodiments, each of the genes differs from any other gene by at least 2base pairs in the gene library. In some embodiments, at least 90% of thedeliverable genes are error free. In some embodiments, the deliverablegenes comprises an error rate of less than 1/3000 resulting in thegeneration of a sequence that deviates from the sequence of a gene inthe list of genes. In some embodiments, at least 90% of the deliverablegenes comprise an error rate of less than 1 in 3000 bp resulting in thegeneration of a sequence that deviates from the sequence of a gene inthe list of genes. In some embodiments, genes in a subset of thedeliverable genes are covalently linked together. In some embodiments, afirst subset of the list of genes encode for components of a firstmetabolic pathway with one or more metabolic end products. In someembodiments, the method of constructing a gene library further comprisesselecting of the one or more metabolic end products, therebyconstructing the list of genes. In some embodiments, the one or moremetabolic end products comprise a biofuel. In some embodiments, a secondsubset of the list of genes encode for components of a second metabolicpathway with one or more metabolic end products. It is noted that any ofthe embodiments described herein can be combined with any of themethods, devices or systems provided in the current invention.

In practicing any of the methods of constructing a gene library asprovided herein, in some embodiments, the list of genes comprises atleast 500 genes. In some embodiments, the list comprises at least 5000genes. In some embodiments, the list comprises at least 10000 genes. Insome embodiments, the genes are at least 1 kb. In some embodiments, thegenes are at least 2 kb. In some embodiments, the genes are at least 3kb. In some embodiments, the second timepoint as described in themethods of constructing a gene library is less than 25 days apart fromthe first timepoint. In some embodiments, the second timepoint is lessthan 5 days apart from the first timepoint. In some embodiments, thesecond timepoint is less than 2 days apart from the first timepoint. Itis noted that any of the embodiments described herein can be combinedwith any of the methods, devices or systems provided in the currentinvention.

In another aspect, the present invention also provides a method ofsynthesizing n-mer oligonucleotides on a substrate. The method comprisesa) providing a substrate with resolved loci that are functionalized witha chemical moiety suitable for nucleotide coupling; and b) coupling atleast two building blocks to a plurality of growing oligonucleotidechains each residing on one of the resolved loci at a rate of at least12 nucleotides per hour according to a locus specific predeterminedsequence, thereby synthesizing a plurality of oligonucleotides that aren basepairs long. Various embodiments related to the method ofsynthesizing n-mer oligonucleotides on a substrate are described herein.

In any of the methods of synthesizing n-mer oligonucleotides on asubstrate as provided herein, in some embodiments, the methods furthercomprise coupling at least two building blocks to a plurality of growingoligonucleotide chains each residing on one of the resolved loci at arate of at least 15 nucleotides per hour. In some embodiments, themethod further comprises coupling at least two building blocks to aplurality of growing oligonucleotide chains each residing on one of theresolved loci at a rate of at least 20 nucleotides per hour. In someembodiments, the method further comprises coupling at least two buildingblocks to a plurality of growing oligonucleotide chains each residing onone of the resolved loci at a rate of at least 25 nucleotides per hour.In some embodiments, at least one resolved locus comprises n-meroligonucleotides deviating from the locus specific predeterminedsequence with an error rate of less than 1/500 bp. In some embodiments,at least one resolved locus comprises n-mer oligonucleotides deviatingfrom the locus specific predetermined sequence with an error rate ofless than 1/1000 bp. In some embodiments, at least one resolved locuscomprises n-mer oligonucleotides deviating from the locus specificpredetermined sequence with an error rate of less than 1/2000 bp. Insome embodiments, the plurality of oligonucleotides on the substratedeviate from respective locus specific predetermined sequences at anerror rate of less than 1/500 bp. In some embodiments, the plurality ofoligonucleotides on the substrate deviate from respective locus specificpredetermined sequences at an error rate of less than 1/1000 bp. In someembodiments, the plurality of oligonucleotides on the substrate deviatefrom respective locus specific predetermined sequences at an error rateof less than 1/2000 bp.

In practicing any of the methods of synthesizing n-mer oligonucleotideson a substrate as provided herein, in some embodiments, the buildingblocks comprise an adenine, guanine, thymine, cytosine, or uridinegroup, or a modified nucleotide. In some embodiments, the buildingblocks comprise a modified nucleotide. In some embodiments, the buildingblocks comprise dinucleotides or trinucleotides. In some embodiments,the building blocks comprise phosphoramidite. In some embodiments, n ofthe n-mer oligonucleotides is at least 100. In some embodiments, n is atleast 200. In some embodiments, n is at least 300. In some embodiments,n is at least 400. In some embodiments, the surface comprises at least100,000 resolved loci and at least two of the plurality of growingoligonucleotides can be different from each other.

In some embodiments, the method of synthesizing n-mer oligonucleotideson a substrate as described herein further comprises vacuum drying thesubstrate before coupling. In some embodiments, the building blockscomprise a blocking group. In some embodiments, the blocking groupcomprises an acid-labile DMT. In some embodiments, the acid-labile DMTcomprises 4,4′-dimethoxytrityl. In some embodiments, the method ofsynthesizing n-mer oligonucleotides on a substrate as described hereinfurther comprises oxidation or sulfurization. In some embodiments, themethod of synthesizing n-mer oligonucleotides on a substrate asdescribed herein further comprises chemically capping uncoupledoligonucleotide chains. In some embodiments, the method of synthesizingn-mer oligonucleotides on a substrate as described herein furthercomprises removing the blocking group, thereby deblocking the growingoligonucleotide chain. In some embodiments, the position of thesubstrate during the coupling step is within 10 cm of the position ofthe substrate during the vacuum drying step. In some embodiments, theposition of the substrate during the coupling step is within 10 cm ofthe position of the substrate during the oxidation step. In someembodiments, the position of the substrate during the coupling step iswithin 10 cm of the position of the substrate during the capping step.In some embodiments, the position of the substrate during the couplingstep is within 10 cm of the position of the substrate during thedeblocking step. In some embodiments, the substrate comprises at least10,000 vias providing fluid communication between a first surface of thesubstrate and a second surface of the substrate. In some embodiments,the substrate comprises at least 100,000 vias providing fluidcommunication between a first surface of the substrate and a secondsurface of the substrate. In some embodiments, the substrate comprisesat least 1,000,000 vias providing fluid communication between a firstsurface of the substrate and a second surface of the substrate. It isnoted that any of the embodiments described herein can be combined withany of the methods, devices or systems provided in the currentinvention.

In another aspect of the present invention, a system for conducting aset of parallel reactions is provided herein. The system comprises: afirst surface with a plurality of resolved loci; a capping element witha plurality of resolved reactor caps. In some embodiments, the systemaligns the plurality of resolved reactor caps with the plurality ofresolved loci on the first surface forming a temporary seal between thefirst surface and the capping element, thereby physically dividing theloci on the first surface into groups of at least two loci into areactor associated with each reactor cap. In some embodiments, eachreactor holds a first set of reagents.

In some embodiments related to any of the systems for conducting a setof parallel reactions as described herein, upon release from the firstsurface, the reactor caps retain at least a portion of the first set ofreagents. In some embodiments, the portion is about 30%. In someembodiments, the portion is about 90%. In some embodiments, theplurality of resolved loci resides on microstructures fabricated into asupport surface. In some embodiments, the plurality of resolved loci isat a density of at least 1 per mm². In some embodiments, the pluralityof resolved loci is at a density of at least 10 per mm². In someembodiments, the plurality of resolved loci are at a density of at least100 per mm². In some embodiments, the microstructures comprise at leasttwo channels in fluidic communication with each other. In someembodiments, the at least two channels comprise two channels withdifferent width. In some embodiments, at least two channels comprise twochannels with different length. In some embodiments, at least one of thechannels is longer than 100 μm. In some embodiments, at least one of thechannels is shorter than 1000 μm. In some embodiments, at least one ofthe channels is wider than 50 μm in diameter. In some embodiments, atleast one of the channels is narrower than 100 μm in diameter. In someembodiments, the system further comprises a second surface with aplurality of resolved loci at a density of at least 0.1 per mm². In someembodiments, the system further comprises a second surface with aplurality of resolved loci at a density of at least 1 per mm². In someembodiments, the system further comprises a second surface with aplurality of resolved loci at a density of at least 10 per mm².

In some embodiments related to any of the systems for conducting a setof parallel reactions as described herein, the resolved loci of thefirst surface comprise a coating of reagents. In some embodiments, theresolved loci of the second surface comprise a coating of reagents. Insome embodiments, the coating of reagents is covalently linked to thefirst or second surface. In some embodiments, the coating of reagentscomprises oligonucleotides. In some embodiments, the coating of reagentshas a surface area of at least 1.45 μm² per 1.0 μm² of planar surfacearea. In some embodiments, the coating of reagents has a surface area ofat least 1.25 μm² per 1.0 μm² of planar surface area. In someembodiments, the coating of reagents has a surface area of at least 1μm² per 1.0 μm² of planar surface area. In some embodiments, theresolved loci in the plurality of resolved loci comprise a nominalarclength of the perimeter at a density of at least 0.001 μm/μm². Insome embodiments, the resolved loci in the plurality of resolved locicomprise a nominal arclength of the perimeter at a density of at least0.01 μm/μm². In some embodiments, the resolved loci in the plurality ofresolved loci of the first surface comprise a high energy surface. Insome embodiments, the first and second surfaces comprise a differentsurface tension with a given liquid. In some embodiments, the highsurface energy corresponds to a water contact angle of less than 20degree. In some embodiments, the plurality of resolved loci are locatedon a solid substrate comprising a material selected from the groupconsisting of silicon, polystyrene, agarose, dextran, cellulosicpolymers, polyacrylamides, PDMS, and glass. In some embodiments, thecapping elements comprise a material selected from the group consistingof silicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, PDMS, and glass. It is noted that any of theembodiments described herein can be combined with any of the methods,devices or systems provided in the current invention.

In yet another aspect, the present invention also provides an array ofenclosures. The array of enclosures comprise: a plurality of resolvedreactors comprising a first substrate and a second substrate comprisingreactor caps; at least 2 resolved loci in each reactor. In some cases,the resolved reactors are separated with a releasable seal. In somecases, the reactor caps retain at least a part of the contents of thereactors upon release of the second substrate from the first substrate.In some embodiments, the reactor caps on the second substrate have adensity of at least 0.1 per mm². In some embodiments, reactor caps onthe second substrate have a density of at least 1 per mm². In someembodiments, reactor caps on the second substrate have a density of atleast 10 per mm².

In some embodiments related to the array of enclosures as providedherein, the reactor caps retain at least 30% of the contents of thereactors. In some embodiments, the reactor caps retain at least 90% ofthe contents of the reactors. In some embodiments, the resolved loci areat a density of at least 2/mm². In some embodiments, the resolved lociare at a density of at least 100/mm². In some embodiments, the array ofenclosures further comprises at least 5 resolved loci in each reactor.In some embodiments, the array of enclosures as described herein furthercomprises at least 20 resolved loci in each reactor. In someembodiments, the array of enclosures as described herein furthercomprises at least 50 resolved loci in each reactor. In someembodiments, the array of enclosures as described herein furthercomprises at least 100 resolved loci in each reactor.

In some embodiments related to the array of enclosures as describedherein, the resolved loci reside on microstructures fabricated into asupport surface. In some embodiments, the microstructures comprise atleast two channels in fluidic communication with each other. In someembodiments, the at least two channels comprise two channels withdifferent width. In some embodiments, the at least two channels comprisetwo channels with different length. In some embodiments, at least one ofthe channels is longer than 100 μm. In some embodiments, at least one ofthe channels is shorter than 1000 μm. In some embodiments, at least oneof the channels is wider than 50 μm in diameter. In some embodiments, atleast one of the channels is narrower than 100 μm in diameter. In someembodiments, the microstructures comprise a nominal arclength of theperimeter of the at least two channels that has a density of at least0.01 μm/square μm. In some embodiments, the microstructures comprise anominal arclength of the perimeter of the at least two channels that hasa density of at least 0.001 μm/square μm. In some embodiments, theresolved reactors are separated with a releasable seal. In someembodiments, the seal comprises a capillary burst valve.

In some embodiments related to the array of enclosures as describedherein, the plurality of resolved loci of the first substrate comprise acoating of reagents. In some embodiments, the plurality of resolved lociof the second substrate comprises a coating of reagents. In someembodiments, the coating of reagents is covalently linked to the firstor second surface. In some embodiments, the coating of reagentscomprises oligonucleotides. In some embodiments, the coating of reagentshas a surface area of at least 1 μm² per 1.0 μm² of planar surface area.In some embodiments, the coating of reagents has a surface area of atleast 1.25 μm² per 1.0 μm² of planar surface area. In some embodiments,the coating of reagents has a surface area of at least 1.45 μm² per 1.0μm² of planar surface area. In some embodiments, the plurality ofresolved loci of the first substrate comprises a high energy surface. Insome embodiments, the first and second substrates comprise a differentsurface tension with a given liquid. In some embodiments, the surfaceenergy corresponds to a water contact angle of less than 20 degree. Insome embodiments, the plurality of resolved loci or the reactor caps arelocated on a solid substrate comprising a material selected from thegroup consisting of silicon, polystyrene, agarose, dextran, cellulosicpolymers, polyacrylamides, PDMS, and glass. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays or systems provided in the current invention.

In still yet another aspect, the present invention also provides amethod of conducting a set of parallel reactions. The method comprises:(a) providing a first surface with a plurality of resolved loci; (b)providing a capping element with a plurality of resolved reactor caps;(c) aligning the plurality of resolved reactor caps with the pluralityof resolved loci on the first surface and forming a temporary sealbetween the first surface and the capping element, thereby physicallydividing the loci on the first surface into groups of at least two loci;(d) performing a first reaction, thereby forming a first set ofreagents; and (e) releasing the capping element from the first surface,wherein each reactor cap retains at least a portion of the first set ofreagents in a first reaction volume. In some embodiments, the portion isabout 30%. In some embodiments, the portion is about 90%.

In some embodiments, the method of conducting a set of parallelreactions as described herein further comprises the steps of: (f)providing a second surface with a plurality of resolved loci; (g)aligning the plurality of resolved reactor caps with the plurality ofresolved loci on the second surface and forming a temporary seal betweenthe second surface and the capping element, thereby physically dividingthe loci on the second surface; (h) performing a second reaction usingthe portion of the first set of reagents, thereby forming a second setof reagents; and (i) releasing the capping element from the secondsurface, wherein each reactor cap can retain at least a portion of thesecond set of reagents in a second reaction volume. In some embodiments,the portion is about 30%. In some embodiments, the portion is about 90%.

In practicing any of the methods of conducting a set of parallelreactions as described herein, the plurality of resolved loci can have adensity of at least 1 per mm² on the first surface. In some embodiments,the plurality of resolved loci have a density of at least 10 per mm² onthe first surface. In some embodiments, the plurality of resolved locihave a density of at least 100 per mm² on the first surface. In someembodiments, the plurality of resolved reactor caps have a density of atleast 0.1 per mm² on the capping element. In some embodiments, theplurality of resolved reactor caps have a density of at least 1 per mm²on the capping element. In some embodiments, the plurality of resolvedreactor caps have a density of at least 10 per mm² on the cappingelement. In some embodiments, the plurality of resolved loci have adensity of more than 0.1 per mm² on the second surface. In someembodiments, the plurality of resolved loci have a density of more than1 per mm² on the second surface. In some embodiments, the plurality ofresolved loci have a density of more than 10 per mm² on the secondsurface.

In practicing any of the methods of conducting a set of parallelreactions as described herein, the releasing of the capping elementsfrom the surface steps such as the releasing steps in (e) and (i) asdescribed herein can be performed at a different velocity. In someembodiments, the resolved loci of the first surface comprise a coatingof reagents for the first reaction. In some embodiments, the resolvedloci of the second surface comprise a coating of reagents for the secondreaction. In some embodiments, the coating of reagents is covalentlylinked to the first or second surface. In some embodiments, the coatingof reagents comprises oligonucleotides. In some embodiments, the coatingof reagents has a surface area of at least 1 μm² per 1.0 μm² of planarsurface area. In some embodiments, the coating of reagents has a surfacearea of at least 1.25 μm² per 1.0 μm² of planar surface area. In someembodiments, the coating of reagents has a surface area of at least 1.45μm² per 1.0 μm² of planar surface area. In some embodiments, theoligonucleotides are at least 25 bp. In some embodiments, theoligonucleotides are at least 200 bp. In some embodiments, theoligonucleotides are at least 300 bp. In some embodiments, the resolvedloci of the first surface comprise a high energy surface. In someembodiments, the first and second surfaces comprise a different surfacetension with a given liquid. In some embodiments, the surface energycorresponds to a water contact angle of less than 20 degree.

In some embodiments related to the method of conducting a set ofparallel reactions as described herein, the plurality of resolved locior the resolved reactor caps are located on a solid substrate comprisinga material selected from the group consisting of silicon, polystyrene,agarose, dextran, cellulosic polymers, polyacrylamides, PDMS, and glass.In some embodiments, the first and second reaction volumes aredifferent. In some embodiments, the first or second reaction comprisespolymerase cycling assembly. In some embodiments, the first or secondreaction comprises enzymatic gene synthesis, annealing and ligationreaction, simultaneous synthesis of two genes via a hybrid gene, shotgunligation and co-ligation, insertion gene synthesis, gene synthesis viaone strand of DNA, template-directed ligation, ligase chain reaction,microarray-mediated gene synthesis, solid-phase assembly, Sloningbuilding block technology, or RNA ligation mediated gene synthesis. Insome embodiments, the methods of conducting a set of parallel reactionsas described herein further comprises cooling the capping element. Insome embodiments, the method of conducting a set of parallel reactionsas described herein further comprises cooling the first surface. In someembodiments, the method of conducting a set of parallel reactions asdescribed herein further comprises cooling the second surface. It isnoted that any of the embodiments described herein can be combined withany of the methods, devices, arrays or systems provided in the currentinvention.

In another aspect, the present invention provides a substrate having afunctionalized surface. The substrate having a functionalized surfacecan comprise a solid support having a plurality of resolved loci. Insome embodiments, the resolved loci are functionalized with a moietythat increases the surface energy of the solid support. In someembodiments, the resolved loci are localized on microchannels.

In some embodiments related to the substrate having a functionalizedsurface as described herein, the moiety is a chemically inert moiety. Insome embodiments, the microchannels comprise a volume of less than 1 nl.In some embodiments, the microchannels comprise a density of the nominalarclength of the perimeter of 0.036 μm/square μm. In some embodiments,the functionalized surface comprises a nominal surface area of at least1 μm² per 1.0 μm² of planar surface area of the substrate. In someembodiments, the functionalized surface comprises a nominal surface areaof at least 1.25 μm² per 1.0 μm² of planar surface area of thesubstrate. In some embodiments, the functionalized surface comprises anominal surface area of at least 1.45 μm² per 1.0 μm² of planar surfacearea of the substrate. In some embodiments, the resolved loci in theplurality of resolved loci comprise a coating of reagents. In someembodiments, the coating of reagents is covalently linked to thesubstrate. In some embodiments, the coating of reagents comprisesoligonucleotides. In some embodiments, at least one of the microchannelsis longer than 100 μm. In some embodiments, at least one of themicrochannels is shorter than 1000 μm. In some embodiments, at least oneof the microchannels is wider than 50 μm in diameter. In someembodiments, at least one of the microchannels is narrower than 100 μmin diameter. In some embodiments, the surface energy corresponds to awater contact angle of less than 20 degree. In some embodiments, thesolid support comprises a material selected from the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, PDMS, and glass. In some embodiments, the plurality ofresolved loci is at a density of at least 1/mm². In some embodiments,the plurality of resolved loci is at a density of at least 100/mm². Itis noted that any of the embodiments described herein can be combinedwith any of the methods, devices, arrays, substrates or systems providedin the current invention.

In another aspect, the present invention also provides a method forsynthesizing oligonucleotides on a substrate having a functionalizedsurface. The method comprises: (a) applying through at least one inkjetpump at least one drop of a first reagent to a first locus of aplurality of loci; (b) applying negative pressure to the substrate; and(c) applying through at least one inkjet pump at least one drop of asecond reagent to the first locus.

In practicing any of the methods for synthesizing oligonucleotides on asubstrate having a functionalized surface as described herein, the firstand second reagents can be different. In some embodiments, the firstlocus is functionalized with a moiety that increases their surfaceenergy. In some embodiments, the moiety is a chemically inert moiety. Insome embodiments, the plurality of loci resides on microstructuresfabricated into the substrate surface. In some embodiments, themicrostructures comprise at least two channels in fluidic communicationwith each other. In some embodiments, the at least two channels comprisetwo channels with different width. In some embodiments, the at least twochannels comprise two channels with different length. In someembodiments, at least one of the channels is longer than 100 μm. In someembodiments, at least one of the channels is shorter than 1000 μm. Insome embodiments, at least one of the channels is wider than 50 μm indiameter. In some embodiments, at least one of the channels is narrowerthan 100 μm in diameter. In some embodiments, the substrate surfacecomprises a material selected from the group consisting of silicon,polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides,PDMS, and glass.

In some embodiments related to the methods for synthesizingoligonucleotides on a substrate having a functionalized surface asdescribed herein, the volume of the drop of the first and/or the secondreagents is at least 2 pl. In some embodiments, the volume of the dropis about 40 pl. In some embodiments, the volume of the drop is at most100 pl. In some embodiments, the microchannels comprise a density of thenominal arclength of the perimeter of at least 0.01 μm/μm². In someembodiments, the microchannels comprise a density of the nominalarclength of the perimeter of at least 0.001 μm/μm². In someembodiments, the functionalized surface comprises a nominal surface areaof at least 1 μm² per 1.0 μm² of planar surface area of the substrate.In some embodiments, the functionalized surface comprises a nominalsurface area of at least 1.25 μm² per 1.0 μm² of planar surface area ofthe substrate. In some embodiments, the functionalized surface comprisesa nominal surface area of at least 1.45 μm² per 1.0 μm² of planarsurface area of the substrate. In some embodiments, the pressuresurrounding the substrate is reduced to less than 1 mTorr. It is notedthat any of the embodiments described herein can be combined with any ofthe methods, devices, arrays, substrates or systems provided in thecurrent invention.

In some embodiments, the method for synthesizing oligonucleotides on asubstrate having a functionalized surface as described herein furthercomprises coupling at least a first building block originating from thefirst drop to a growing oligonucleotide chain on the first locus. Insome embodiments, the building blocks comprise a blocking group. In someembodiments, the blocking group comprises an acid-labile DMT. In someembodiments, the acid-labile DMT comprises 4,4′-dimethoxytrityl. In someembodiments, the method for synthesizing oligonucleotides on a substratehaving a functionalized surface as described herein further comprisesoxidation or sulfurization. In some embodiments, the method forsynthesizing oligonucleotides on a substrate having a functionalizedsurface as described herein further comprises chemically cappinguncoupled oligonucleotide chains. In some embodiments, the method forsynthesizing oligonucleotides on a substrate having a functionalizedsurface as described herein further comprises removing the blockinggroup, thereby deblocking the growing oligonucleotide chain. In someembodiments, the position of the substrate during the negative pressureapplication is within 10 cm of the position of the substrate during thecoupling step. In some embodiments, the position of the substrate duringthe negative pressure application is within 10 cm of the position of thesubstrate during the oxidation step. In some embodiments, the positionof the substrate during the negative pressure application is within 10cm of the position of the substrate during the capping step. In someembodiments, the position of the substrate during the negative pressureapplication is within 10 cm of the position of the substrate during thedeblocking step. In some embodiments, the first locus resides on amicrostructure fabricated into the substrate surface. In someembodiments, at least one reagent for the oxidation step is provided byflooding the microstructure with a solution comprising the at least onereagent. In some embodiments, at least one reagent for the capping stepis provided by flooding the microstructure with a solution comprisingthe at least one reagent. In some embodiments, the first locus resideson a microstructure fabricated into the substrate surface and at leastone reagent for the deblocking step can be provided by flooding themicrostructure with a solution comprising the at least one reagent. Insome embodiments, the method for synthesizing oligonucleotides on asubstrate having a functionalized surface as described herein furthercomprises enclosing the substrate within a sealed chamber. In someembodiments, the sealed chamber allows for purging of liquids from thefirst locus. In some embodiments, the method for synthesizingoligonucleotides on a substrate having a functionalized surface asdescribed herein further comprises draining a liquid through a drainthat is operably linked to the first locus. In some embodiments, afterapplying the negative pressure to the substrate, the moisture content onthe substrate is less than 1 ppm. In some embodiments, the surfaceenergy is increased corresponding to a water contact angle of less than20 degree. It is noted that any of the embodiments described herein canbe combined with any of the methods, devices, arrays, substrates orsystems provided in the current invention.

In yet another aspect, the present invention provides a method ofdepositing reagents to a plurality of resolved loci. The methodcomprises applying through an inkjet pump at least one drop of a firstreagent to a first locus of the plurality of loci; applying through aninkjet pump at least one drop of a second reagent to a second locus ofthe plurality of resolved loci. In some embodiments, the second locus isadjacent to the first locus. In still some embodiments, the first andsecond reagents are different. In still yet some embodiments, the firstand second loci reside on microstructures fabricated into a supportsurface. In yet some embodiments, the microstructures comprise at leastone channel that is more than 100 μm deep.

In practicing any of the methods of depositing reagents to a pluralityof resolved loci as described herein, in some embodiments, themicrostructures comprise at least two channels in fluidic communicationwith each other. In some embodiments, the at least two channels comprisetwo channels with different width. In some embodiments, the at least twochannels comprise two channels with different length. In someembodiments, the first locus receives less than 0.1% of the secondreagent and the second locus receives less than 0.1% of the firstreagent. In some embodiments, the loci comprise a density of the nominalarclength of the perimeter of at least 0.01 μm/square μm. In someembodiments, the loci comprise a density of the nominal arclength of theperimeter of at least 0.001 μm/square μm. In some embodiments, the firstand second loci comprise a coating of reagents. In some embodiments, thecoating of reagents is covalently linked to the substrate. In someembodiments, the coating of reagents comprises oligonucleotides. In someembodiments, at least one of the channels is longer than 100 μm. In someembodiments, at least one of the channels is shorter than 1000 μm. Insome embodiments, at least one of the channels is wider than 50 μm indiameter. In some embodiments, at least one of the channels is narrowerthan 100 μm in diameter. In some embodiments, the support surfacecomprises a material selected from the group consisting of silicon,polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides,PDMS, and glass. In some embodiments, the plurality of resolved loci isat a density of at least 1/mm². In some embodiments, the plurality ofresolved loci is at a density of at least 100/mm². In some embodiments,the volume of the drop is at least 2 pl. In some embodiments, the volumeof the drop is about 40 pl. In some embodiments, the volume of the dropis at most 100 pl. It is noted that any of the embodiments describedherein can be combined with any of the methods, devices, arrays,substrates or systems provided in the current invention.

In still yet another aspect, the present invention provides amicrofluidic system. The microfluidic system comprises a first surfacewith a plurality of microwells at a density of at least 10 per mm²; anda droplet inside one of the plurality of microwells. In someembodiments, the droplet inside one of the plurality of microwells has aReynolds number at a range of about 1-1000. In some embodiments, theplurality of microwells is at a density of at least 1 per mm². In someembodiments, plurality of microwells is at a density of at least 10 permm².

In some embodiments related to the microfluidic system as providedherein, the microfluidic system further comprises an inkjet pump. Insome embodiments, the droplet is deposited by the inkjet pump. In someembodiments, the droplet is moving in the lower half of a firstmicrowell dimension. In some embodiments, the droplet is moving in themiddle third of a first microwell dimension. In some embodiments, theplurality of microwells is at a density of at least 100 per mm². In someembodiments, the first microwell dimension is larger than the droplet.In some embodiments, the microwell is longer than 100 μm. In someembodiments, the microwell is shorter than 1000 μm. In some embodiments,the microwell is wider than 50 μm in diameter. In some embodiments, themicrowell is narrower than 100 μm in diameter. In some embodiments, thevolume of the droplet is at least 2 pl. In some embodiments, the volumeof the droplet is about 40 pl. In some embodiments, the volume of thedroplet is at most 100 pl. In some embodiments, each of the plurality ofmicrowells is fluidically connected to at least one microchannel. Insome embodiments, the at least one microchannel is coated with a moietythat increases surface energy. In some embodiments, the moiety is achemically inert moiety. In some embodiments, the surface energycorresponds to a water contact angle of less than 20 degree. In someembodiments, the microwells are formed on a solid support comprising amaterial selected from the group consisting of silicon, polystyrene,agarose, dextran, cellulosic polymers, polyacrylamides, PDMS, and glass.In some embodiments, the microchannels comprise a density of the nominalarclength of the perimeter of at least 0.01 μm/square μm. In someembodiments, the microchannels comprise a density of the nominalarclength of the perimeter of 0.001 μm/μm². In some embodiments, thesurface coated with the moiety comprises a nominal surface area of atleast 1 μm² per 1.0 μm² of planar surface area of the first surface. Insome embodiments, the surface coated with the moiety comprises a nominalsurface area of at least 1.25 μm² per 1.0 μm² of planar surface area ofthe first surface. In some embodiments, the surface coated with themoiety comprises a nominal surface area of at least 1.45 μm² per 1.0 μm²of planar surface area of the first surface. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays, substrates or systems provided in the currentinvention. In some embodiments, the droplet comprises a reagent thatenables oligonucleotide synthesis. In some embodiments, the reagent is anucleotide or nucleotide analog.

In another aspect, the present invention provides a method of depositingdroplets to a plurality of microwells. The method comprises applyingthrough an inkjet pump at least one droplet to a first microwell of theplurality of microwells. In some cases, the droplet inside one of theplurality of microwells has a Reynolds number at a range of about1-1000. In some embodiments, the plurality of microwells has a densityof at least 1/mm². In yet some cases, the plurality of microwells has adensity of at least 10/mm².

In practicing any of the methods of depositing droplets to a pluralityof microwells as provided herein, the plurality of microwells can have adensity of at least 100/mm². In some embodiments, the microwell islonger than 100 μm. In some embodiments, the microwell is shorter than1000 μm. In some embodiments, the microwell is wider than 50 μm indiameter. In some embodiments, the microwell is narrower than 100 μm indiameter. In some embodiments, the droplet is applied at a velocity ofat least 2 m/sec. In some embodiments, the volume of the droplet is atleast 2 pl. In some embodiments, the volume of the droplet is about 40pl. In some embodiments, the volume of the droplet is at most 100 pl. Insome embodiments, each of the plurality of microwells is fluidicallyconnected to at least one microchannel. In some embodiments, the atleast one microwell is coated with a moiety that increases surfaceenergy. In some embodiments, the moiety is a chemically inert moiety. Insome embodiments, the surface energy corresponds to a water contactangle of less than 20 degree. In some embodiments, the microwells areformed on a solid support comprising a material selected from the groupconsisting of silicon, polystyrene, agarose, dextran, cellulosicpolymers, polyacrylamides, PDMS, and glass. In some embodiments, themicrochannels comprise a density of the nominal arclength of theperimeter of at least 0.01 μm/square μm. In some embodiments, themicrochannels comprise a density of the nominal arclength of theperimeter of at least 0.001 μm²m/μm². In some embodiments, the surfacecoated with the moiety comprises a nominal surface area of at least 1μm² per 1.0 μm² of planar surface area of the first surface. In someembodiments, the surface coated with the moiety comprises a nominalsurface area of at least 1.25 μm² per 1.0 μm² of planar surface area ofthe first surface. In some embodiments, the surface coated with themoiety comprises a nominal surface area of at least 1.45 μm² per 1.0 μm²of planar surface area of the first surface. In some embodiments, adroplet inside a microwell is traveling in the middle third of themicrowell. In some embodiments, a droplet inside a microwell istraveling in the bottom half of the microwell. In some embodiments,droplet comprises a reagent that enables oligonucleotide synthesis. Insome embodiments, the reagent is a nucleotide or nucleotide analog. Itis noted that any of the embodiments described herein can be combinedwith any of the methods, devices, arrays, substrates or systems providedin the current invention.

In another aspect, the present invention also provides a method ofpartitioning. The method of partitioning comprises contacting a firstsurface comprising a liquid at a first plurality of resolved loci with asecond surface comprising a second plurality of resolved loci;determining a velocity of release such that a desired fraction of theliquid can be transferred from the first plurality of resolved loci tothe second plurality of resolved loci; and detaching the second surfacefrom the first surface at said velocity. In some embodiments, the firstsurface comprises a first surface tension with the liquid, and thesecond surface can comprise a second surface tension with the liquid.

In practicing any of the methods of partitioning as provided herein, aportion of the first surface can be coated with a moiety that increasessurface tension. In some embodiments, the moiety is a chemically inertmoiety. In some embodiments, the surface tension of the first surfacecorresponds to a water contact angle of less than 20 degree. In someembodiments, the surface tension of the second surface corresponds to awater contact angle of more than 90 degree. In some embodiments, thefirst surface comprises a material selected from the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, PDMS, and glass. In some embodiments, the plurality ofresolved loci comprises a density of the nominal arclength of theperimeter of at least 0.01 μm/μm². In some embodiments, the plurality ofresolved loci comprises a density of the nominal arclength of theperimeter of at least 0.001 μm/μm². In some embodiments, the surfacecoated with the moiety comprises a nominal surface area of at least 1μm² per 1.0 μm² of planar surface area of the first surface. In someembodiments, the surface coated with the moiety comprises a nominalsurface area of at least 1.25 μm² per 1.0 μm² of planar surface area ofthe first surface. In some embodiments, the surface coated with themoiety comprises a nominal surface area of at least 1.45 μm² per 1.0 μm²of planar surface area of the first surface. In some embodiments, thefirst plurality of resolved loci is at a density of at least 1/mm². Insome embodiments, the first plurality of resolved loci is at a densityof at least 100/mm². In some embodiments, the first or the secondsurface comprises microchannels holding at least a portion of theliquid. In some embodiments, the first or the second surface comprisesnanoreactors holding at least a portion of the liquid. In someembodiments, the method of partitioning as described herein furthercomprises contacting a third surface with a third plurality of resolvedloci. In some embodiments, the liquid comprises a nucleic acid. In someembodiments, the desired fraction is more than 30%. In some embodiments,the desired fraction is more than 90%. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays, substrates or systems provided in the currentinvention.

In yet another aspect, the present invention also provides a method ofmixing as described herein. The method comprises: (a) providing a firstsubstrate comprising a plurality of microstructures fabricated thereto;(b) providing a second substrate comprising a plurality of resolvedreactor caps; (c) aligning the first and second substrates such that afirst reactor cap of the plurality can be configured to receive liquidfrom n microstructures in the first substrate; and (d) delivering liquidfrom the n microstructures into the first reactor cap, thereby mixingliquid from the n microstructures forming a mixture.

In practicing any of the methods of mixing as described herein, theplurality of resolved reactor caps can be at a density of at least0.1/mm². In some embodiments, the plurality of resolved reactor caps areat a density of at least 1/mm². In some embodiments, plurality ofresolved reactor caps are at a density of at least 10/mm². In someembodiments, each of the plurality of microstructures can comprise atleast two channels of different width. In some embodiments, the at leastone of the channels is longer than 100 μm. In some embodiments, the atleast one of the channels is shorter than 1000 μm. In some embodiments,the at least one of the channels is wider than 50 μm in diameter. Insome embodiments, the at least one of the channels is narrower than 100μm in diameter. In some embodiments, the at least one of the channels iscoated with a moiety that increases surface energy. In some embodiments,the moiety is a chemically inert moiety. In some embodiments, themicrostructures are formed on a solid support comprising a materialselected from the group consisting of silicon, polystyrene, agarose,dextran, cellulosic polymers, polyacrylamides, PDMS, and glass. In someembodiments, the microchannels comprise a density of the nominalarclength of the perimeter of at least 0.01 μm/square μm. In someembodiments, the microchannels comprise a density of the nominalarclength of the perimeter of at least 0.001 μm/m². In some embodiments,the surface coated with the moiety comprises a nominal surface area ofat least 1 μm² per 1.0 μm² of planar surface area of the first surface.In some embodiments, the surface coated with the moiety comprises anominal surface area of at least 1.25 μm² per 1.0 μm² of planar surfacearea of the first surface. In some embodiments, the surface coated withthe moiety comprises a nominal surface area of at least 1.45 μm² per 1.0μm² of planar surface area of the first surface. In some embodiments,the plurality of microstructures comprises a coating of reagents. Insome embodiments, the coating of reagents is covalently linked to thefirst surface. In some embodiments, the coating of reagents comprisesoligonucleotides. In some embodiments, the microstructures are at adensity of at least 1/mm². In some embodiments, the microstructures areat a density of at least 100/mm².

In some embodiments related to the methods of mixing as describedherein, after step (c), which is aligning the first and secondsubstrates such that a first reactor cap of the plurality can beconfigured to receive liquid from n microstructures in the firstsubstrate, there is a gap of less than 100 μm between the first and thesecond substrates. In some embodiments, after step (c), there is a gapof less than 50 μm between the first and the second substrates. In someembodiments, after step (c), there is a gap of less than 20 μm betweenthe first and the second substrates. In some embodiments, after step(c), there is a gap of less than 10 μm between the first and the secondsubstrates. In some embodiments, the mixture partially spreads into thegap. In some embodiments, the method of mixing further comprises sealingthe gap by bringing the first and the second substrate closer together.In some embodiments, one of the two channels is coated with a moietythat increases surface energy corresponding to a water contact angle ofless than 20 degree. In some embodiments, the moiety is a chemicallyinert moiety. In some embodiments, the delivering is performed bypressure. In some embodiments, the volume of the mixture is greater thanthe volume of the reactor cap. In some embodiments, the liquid comprisesa nucleic acid. In some embodiments, n is at least 10. In someembodiments, n is at least 25. In some embodiments, n, the number ofmicrostructures from which the liquid is mixed forming a mixture, can beat least 50. In some embodiments, n is at least 75. In some embodiments,n is at least 100. It is noted that any of the embodiments describedherein can be combined with any of the methods, devices, arrays,substrates or systems provided in the current invention.

In yet another aspect, the present invention also provides a method ofsynthesizing n-mer oligonucleotides on a substrate as described herein.The method comprises: providing a substrate with resolved loci that arefunctionalized with a chemical moiety suitable for nucleotide coupling;and coupling at least two building blocks to a plurality of growingoligonucleotide chains each residing on one of the resolved lociaccording to a locus specific predetermined sequence withouttransporting the substrate between the couplings of the at least twobuilding blocks, thereby synthesizing a plurality of oligonucleotidesthat are n basepairs long.

In practicing any of the methods of synthesizing n-mer oligonucleotideson a substrate as described herein, the method can further comprisecoupling at least two building blocks to a plurality of growingoligonucleotide chains each residing on one of the resolved loci at arate of at least 12 nucleotides per hour. In some embodiments, themethod further comprises coupling at least two building blocks to aplurality of growing oligonucleotide chains each residing on one of theresolved loci at a rate of at least 15 nucleotides per hour. In someembodiments, the method further comprises coupling at least two buildingblocks to a plurality of growing oligonucleotide chains each residing onone of the resolved loci at a rate of at least 20 nucleotides per hour.In some embodiments, the method further comprises coupling at least twobuilding blocks to a plurality of growing oligonucleotide chains eachresiding on one of the resolved loci at a rate of at least 25nucleotides per hour. In some embodiments, at least one resolved locuscomprises n-mer oligonucleotides deviating from the locus specificpredetermined sequence with an error rate of less than 1/500 bp. In someembodiments, at least one resolved locus comprises n-meroligonucleotides deviating from the locus specific predeterminedsequence with an error rate of less than 1/1000 bp. In some embodiments,at least one resolved locus comprises n-mer oligonucleotides deviatingfrom the locus specific predetermined sequence with an error rate ofless than 1/2000 bp. In some embodiments, the plurality ofoligonucleotides on the substrate deviate from respective locus specificpredetermined sequences at an error rate of less than 1/500 bp. In someembodiments, the plurality of oligonucleotides on the substrate deviatefrom respective locus specific predetermined sequences at an error rateof less than 1/1000 bp. In some embodiments, the plurality ofoligonucleotides on the substrate deviate from respective locus specificpredetermined sequences at an error rate of less than 1/2000 bp.

In some embodiments related to the method of synthesizing n-meroligonucleotides on a substrate as described herein, the building blockscomprise an adenine, guanine, thymine, cytosine, or uridine group, or amodified nucleotide. In some embodiments, the building blocks comprise amodified nucleotide. In some embodiments, the building blocks comprisedinucleotides. In some embodiments, the building blocks comprisephosphoramidite. In some embodiments, n is at least 100. In someembodiments, wherein n is at least 200. In some embodiments, n is atleast 300. In some embodiments, n is at least 400. In some embodiments,the substrate comprises at least 100,000 resolved loci and at least twoof the plurality of growing oligonucleotides are different from eachother. In some embodiments, the method further comprise vacuum dryingthe substrate before coupling. In some embodiments, the building blockscomprise a blocking group. In some embodiments, the blocking groupcomprises an acid-labile DMT. In some embodiments, the acid-labile DMTcomprises 4,4′-dimethoxytrityl. In some embodiments, the method furthercomprise oxidation or sulfurization. In some embodiments, the methodfurther comprise chemically capping uncoupled oligonucleotide chains. Insome embodiments, the method further comprise removing the blockinggroup, thereby deblocking the growing oligonucleotide chain. In someembodiments, the substrate comprises at least 10,000 vias providingfluid communication between a first surface of the substrate and asecond surface of the substrate. In some embodiments, the substratecomprises at least 100,000 vias providing fluid communication between afirst surface of the substrate and a second surface of the substrate. Insome embodiments, the substrate comprises at least 1,000,000 viasproviding fluid communication between a first surface of the substrateand a second surface of the substrate. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays, substrates or systems provided in the currentinvention.

In yet another aspect, the present invention also provides a method ofconstructing a gene library as described herein. The method comprises:entering at a first timepoint, in a computer readable non-transientmedium a list of genes, wherein the list comprises at least 100 genesand wherein the genes are at least 500 bp; synthesizing more than 90% ofthe list of genes, thereby constructing a gene library with deliverablegenes; preparing a sequencing library that represents the gene library;obtaining sequence information; selecting at least a subset of thedeliverable genes based on the sequence information; and delivering theselected deliverable genes at a second timepoint, wherein the secondtimepoint is less than a month apart from the first timepoint.

In practicing any of the methods of constructing a gene library asdescribed herein, the sequence information can be obtained vianext-generation sequencing. The sequence information can be obtained bySanger sequencing. In some embodiments, the method further comprisesdelivering at least one gene at a second timepoint. In some embodiments,at least one of the genes differ from any other gene by at least 0.1% inthe gene library. In some embodiments, each of the genes differ from anyother gene by at least 0.1% in the gene library. In some embodiments, atleast one of the genes differ from any other gene by at least 10% in thegene library. In some embodiments, each of the genes differ from anyother gene by at least 10% in the gene library. In some embodiments, atleast one of the genes differ from any other gene by at least 2 basepairs in the gene library. In some embodiments, each of the genes differfrom any other gene by at least 2 base pairs in the gene library. Insome embodiments, at least 90% of the deliverable genes are error free.In some embodiments, the deliverable genes comprise an error rate ofless than 1/3000 resulting in the generation of a sequence that deviatesfrom the sequence of a gene in the list of genes. In some embodiments,at least 90% of the deliverable genes comprise an error rate of lessthan 1 in 3000 bp resulting in the generation of a sequence thatdeviates from the sequence of a gene in the list of genes. In someembodiments, a subset of the deliverable genes are covalently linkedtogether. In some embodiments, a first subset of the list of genesencode for components of a first metabolic pathway with one or moremetabolic end products. In some embodiments, the method furthercomprises selecting of the one or more metabolic end products, therebyconstructing the list of genes. In some embodiments, the one or moremetabolic end products comprise a biofuel. In some embodiments, a secondsubset of the list of genes encode for components of a second metabolicpathway with one or more metabolic end products. In some embodiments,the list comprises at least 500, genes. In some embodiments, the listcomprises at least 5000 genes. In some embodiments, the list comprisesat least 10000 genes. In some embodiments, the genes are at least 1 kb.In some embodiments, the genes are at least 2 kb. In some embodiments,the genes are at least 3 kb. In some embodiments, the second timepointis less than 25 days apart from the first timepoint. In someembodiments, the second timepoint is less than 5 days apart from thefirst timepoint. In some embodiments, the second timepoint is less than2 days apart from the first timepoint. It is noted that any of theembodiments described herein can be combined with any of the methods,devices, arrays, substrates or systems provided in the currentinvention.

Provided herein, in some embodiments, is a microfluidic device fornucleic acid synthesis, comprising a substantially planar substrateportion comprising n groupings of m microfluidic connections betweenopposite surfaces, wherein each one of the n*m microfluidic connectionscomprises a first channel and a second channel, and wherein the firstchannel within each of the n groupings is common to all m microfluidicconnections, wherein the plurality of microfluidic connections span thesubstantially planar substrate portion along the smallest dimension ofthe substrate, and wherein n and m are at least 2. In some embodiments,the second channel is functionalized with a coating that is capable offacilitating the attachment of an oligonucleotide to the device. In someembodiments, the device further comprises a first oligonucleotide thatis attached to the second channels in k of the n groupings. In someembodiments, k is 1. In some embodiments, the device further comprises asecond oligonucleotide that is attached to 1 of the n groupings. In someembodiments, 1 is 1. In some embodiments, the none of the groupings inthe 1 groupings are in the k groupings.

In some embodiments, the oligonucleotide is at least 10 nucleotides, 25nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 125nucleotides, 150 nucleotides, or 200 nucleotides long.

In some embodiments, the first and the second oligonucleotides differ byat least 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or10 nucleotides.

In some embodiments, the n*m microfluidic connections are at most 5 mm,1.5 mm, 1.0 mm, or 0.5 mm long. In some embodiments, the first channelwithin each of the n groupings is at most 5 mm, 1.5 mm, 1.0 mm, or 0.5mm long. In some embodiments, the first channel within each of the ngroupings is at least 0.05 mm, 0.75 mm, 0.1 mm, 0.2 mm, 0.3 mm, or 0.4mm long. In some embodiments, the second channel in each of the n*mmicrofluidic connections is at most 0.2 mm, 0.1 mm, 0.05 mm, 0.04 mm, or0.03 mm long. In some embodiments, the second channel in each of the n*mmicrofluidic connections is at least 0.001 mm, 0.005 mm, 0.01 mm, 0.02mm, or 0.03 mm long. In some embodiments, the cross section of the firstchannel within each of the n groupings is at least 0.01 mm, 0.025 mm,0.05 mm, or 0.075 mm. In some embodiments, the cross section of thefirst channel within each of the n groupings is at most 1 mm, 0.5 mm,0.25 mm, 0.1 mm, or 0.075 mm. In some embodiments, the cross section ofthe second channel in each of the n*m microfluidic connections is atleast 0.001 mm, 0.05 mm, 0.01 mm, 0.015 mm, or 0.02 mm. In someembodiments, the cross section of the second channel in each of the n*mmicrofluidic connections is at most 0.25 mm, 0.125 mm, 0.050 mm, 0.025mm, 0.02 mm. In some embodiments, the standard deviation in the crosssection of the second channels in each of the n*m microfluidicconnections is less than 25%, 20%, 15%, 10%, 5%, or 1% of the mean ofthe cross section. In some embodiments, the variation in the crosssection within at least 90% of the second channels of the n*mmicrofluidic connections is at most 25%, 20%, 15%, 10%, 5%, or 1%.

In some embodiments, n is at least 10, 25, 50, 100, 1000, or 10000. Insome embodiments, m is at least 3, 4, or 5.

In some embodiments, the substrate comprises at least 5%, 10%, 25%, 50%,80%, 90%, 95%, or 99% silicon.

In some embodiments, at least 90% of the second channels of the n*mmicrofluidic connections is functionalized with a moiety that increasessurface energy. In some embodiments, the surface energy is increased toa level corresponding to a water contact angle of less than 75, 50, 30,or 20 degrees.

In some embodiments, the aspect ratio for at least 90% of the secondchannels of the n*m microfluidic connections is less than 1, 0.5, or0.3. In some embodiments, the aspect ratio for at least 90% of the firstchannels in the n groupings is less than 0.5, 0.3, or 0.2.

In some embodiments, the total length of at least 10%, 25%, 50%, 75%,90%, or 95% of the n*m fluidic connections are within 10%, 20%, 30%,40%, 50%, 100%, 200%, 500%, or 1000% of the smallest dimension of thesubstantially planar substrate.

In some embodiments, the substantially planar portion of the device isfabricated from a SOI wafer.

In another aspect, the invention relates to a method of nucleic acidamplification, comprising: (a) providing a sample comprising ncircularized single stranded nucleic acids, each comprising a differenttarget sequence; (b) providing a first adaptor that is hybridizable toat least one adaptor hybridization sequence on m of the n circularizedsingle stranded nucleic acids; (c) providing conditions suitable forextending the first adaptor using the m circularized single strandednucleic acids as a template, thereby generating m single strandedamplicon nucleic acids, wherein each of the m single stranded ampliconnucleic acids comprises a plurality of replicas of the target sequencefrom its template; (d) providing a first auxiliary oligonucleotide thatis hybridizable to the first adaptor; and (e) providing a first agentunder conditions suitable for the first agent to cut the m singlestranded amplicon nucleic acids at a plurality of cutting sites, therebygenerating a plurality of single stranded replicas of the targetsequences in the m circularized single stranded nucleic acids. In someembodiments, n or m is at least 2. In some embodiments, n or m is atleast 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300,400, or 500. In some embodiments, m is less than n. In some embodiments,the sample comprising the n circularized single stranded nucleic acid isformed by providing at least n linear single stranded nucleic acids,each comprising one of the different target sequences and circularizingthe n linear single stranded nucleic acids, thereby generating the ncircularized single stranded nucleic acids. In some embodiments, thefirst adaptor is hybridizable to both ends of the n linear singlestranded nucleic acids concurrently. In some embodiments, the differenttarget sequences in the n linear single stranded nucleic acids areflanked by a first and a second adaptor hybridization sequence. In someembodiments, the at least n linear single stranded nucleic acids aregenerated by de novo oligonucleotide synthesis. In some embodiments, thefirst adaptor hybridization sequence in each of the n linear singlestranded nucleic acids differ by no more than two nucleotide bases. Insome embodiments, the first or the second adaptor hybridization sequenceis at least 5 nucleotides long. In some embodiments, the first or thesecond adaptor hybridization sequence is at most 75, 50, 45, 40, 35, 30,or 25 nucleotides long. In some embodiments, the ends of the n linearsingle stranded nucleic acids pair with adjacent bases on the firstadaptor when the first adaptor is hybridized to the both ends of thelinear single stranded nucleic acid concurrently. In some embodiments,the locations of the plurality of cutting sites are such that theadaptor hybridization sequence is severed from at least 5% of aremainder sequence portion of the m circularized single stranded nucleicacid replicas. In some embodiments, at least 5% of the sequence of the mcircularized single stranded nucleic acid replicas other than the atleast one adaptor hybridization sequence remains uncut. In someembodiments, the locations of the plurality of cutting sites are outsidethe at least one adaptor hybridization sequence. In some embodiments,the locations of the plurality of cutting sites are independent of thetarget sequences. In some embodiments, the locations of the plurality ofcutting sites are determined by at least one sequence element within thesequence of the first adaptor or the first auxiliary oligonucleotide. Insome embodiments, the sequence element comprises a recognition site fora restriction endonuclease. In some embodiments, the first auxiliaryoligonucleotide or the first adaptor oligonucleotide comprises arecognition site for a Type IIS restriction endonuclease. In someembodiments, the recognition sites are at least 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotides away from the cutting sites. In some embodiments,the plurality of cutting sites are at junctures of single and doublestranded nucleic acids. In some embodiments, the double stranded nucleicacids comprise the first adaptor and the first auxiliaryoligonucleotide. In some embodiments, the single stranded nucleic acidsconsists essentially of the m different target sequences. In someembodiments, the m different target sequences have at most 95% pairwisesimilarity. In some embodiments, the m different target sequences haveat most 90% pairwise similarity. In some embodiments, the m differenttarget sequences have at most 80% pairwise similarity. In someembodiments, the m different target sequences have at most 50% pairwisesimilarity. In some embodiments, generating the m single strandedamplicon nucleic acid comprises strand displacement amplification. Insome embodiments, the first auxiliary oligonucleotide comprises anaffinity tag. In some embodiments, the affinity tag comprises biotin orbiotin derivative. In some embodiments, the method further comprisesisolating double stranded nucleic acids from the sample. In someembodiments, the isolating comprises affinity purification,chromatography, or gel purification. In some embodiments, the firstagent comprises a restriction endonuclease. In some embodiments, thefirst agent comprises at least two restriction endonucleases. In someembodiments, the first agent comprises a Type IIS restrictionendonuclease. In some embodiments, the first agent comprises a nickingendonuclease. In some embodiments, the first agent comprises at leasttwo nicking endonucleases. In some embodiments, the first agentcomprises at least one enzyme selected from the group consisting ofMlyI, SchI, AlwI, BccI, BceAI, BsmAI, BsmFI, FokI, HgaI, PleI, SfaNI,BfuAI, BsaI, BspMI, BtgZI, EarI, BspQI, SapI, SgeI, BceFI, BslFI,BsoMAI, Bst71I, FaqI, AceIII, BbvII, BveI, LguI, BfuCI, DpnII, FatI,MboI, MluCI, Sau3AI, Tsp509I, BssKI, PspGI, StyD4I, Tsp45I, AoxI, BscFI,Bsp143I, BssMI, BseENII, BstMBI, Kzo9I, NedII, Sse9I, TasI, TspEI, AjnI,BstSCI, EcoRII, MaeIII, NmuCI, Psp6I, MnlI, BspCNI, BsrI, BtsCI, HphI,HpyAV, MboII, AcuI, BciVI, BmrI, BpmI, BpuEI, BseRI, BsgI, BsmI, BsrDI,BtsI, EciI, MmeI, NmeAIII, Hin4II, TscAI, Bce83I, BmuI, BsbI, BscCI,NlaIII, Hpy99I, TspRI, FaeI, Hin1II, Hsp92II, SetI, TaiI, TscI, TscAI,TseFI, Nb.BsrDI, Nb.BtsI, AspCNI, BscGI, BspNCI, EcoHI, FinI, TsuI,UbaF11I, UnbI, Vpak11AI, BspGI, DrdII, Pfl1108I, UbaPI, Nt.AlwI,Nt.BsmAI, Nt.BstNBI, and Nt.BspQI, and variants thereof. In someembodiments, the first agent comprises essentially the same function,recognizes the same or essentially the same recognition sequence, orcuts at the same or essentially same cutting site, as any of the listedfirst agents and variants. In some embodiments, the at least tworestriction enzymes comprise MlyI and BciVI or BfuCI and MlyI. In someembodiments, the method further comprises (a) partitioning the sampleinto a plurality of fractions; (b) providing at least one fraction witha second adaptor that is hybridizable to at least one adaptorhybridization sequence on k of the n different circularized singlestranded nucleic acids; (c) providing conditions suitable for extendingthe second adaptor using the k circularized single stranded nucleicacids as a template, thereby generating k single stranded ampliconnucleic acids, wherein the second single stranded amplicon nucleic acidcomprises a plurality of replicas of the target sequence from itstemplate; (d) providing a second auxiliary oligonucleotide that ishybridizable to the second adaptor; and (e) providing a second agentunder conditions suitable for the agent to cut the k single strandedamplicon nucleic acids at a second plurality of cutting sites, therebygenerating a plurality of single stranded replicas of the targetsequences in the k circularized single stranded nucleic acids. In someembodiments, the first and the second adaptors are the same. In someembodiments, the first and the second auxiliary oligonucleotides are thesame. In some embodiments, the first and the second agents are the same.In some embodiments, k+m is less than n. In some embodiments, k is atleast 2. In some embodiments, the sample comprising the n circularizedsingle stranded nucleic acid is formed by single stranded nucleic acidamplification. In some embodiments, the single stranded nucleic acidamplification comprises: (a) providing a sample comprising at least mcircularized single stranded precursor nucleic acids; (b) providing afirst precursor adaptor that is hybridizable to the m circularizedsingle stranded precursor nucleic acids; (c) providing conditionssuitable for extending the first precursor adaptor using the mcircularized single stranded precursor nucleic acids as a template,thereby generating m single stranded precursor amplicon nucleic acids,wherein the single stranded amplicon nucleic acid comprises a pluralityof replicas of the m circularized single stranded precursor nucleicacid; (d) providing a first precursor auxiliary oligonucleotide that ishybridizable to the first precursor adaptor; and (e) providing a firstprecursor agent under conditions suitable for the first precursor agentto cut the first single stranded precursor amplicon nucleic acid at aplurality of cutting sites, thereby generating the m linear precursornucleic acids. In some embodiments, the method further comprisescircularizing the m linear precursor nucleic acids, thereby formingreplicas of the m circularized single stranded precursor nucleic acids.In some embodiments, the m circularized single stranded precursornucleic acid is amplified by at least 10, 100, 250, 500, 750, 1000,1500, 2000, 3000, 4000, 5000, 10000-fold, or more in single strandedreplicas. In some embodiments, at least one of the m circularized singlestranded nucleic acids is at a concentration of about or at most about100 nM, 10 nM, 1 nM, 50 pM, 1 pM, 100 fM, 10 fM, 1 fM, or less. In someembodiments, circularizing comprises ligation. In some embodiments,ligation comprises the use of a ligase selected from the groupconsisting of T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, E. coli DNAligase, Taq DNA ligase, and 9N DNA ligase.

In yet a further aspect, the invention, in various embodiments relatesto a kit comprising: (a) a first adaptor; (b) a first auxiliaryoligonucleotide that is hybridizable to the adaptor; (c) a ligase; and(d) a first cleaving agent, comprising at least one enzyme selected fromthe group consisting of MlyI, SchI, AlwI, BccI, BceAI, BsmAI, BsmFI,FokI, HgaI, PleI, SfaNI, BfuAI, BsaI, BspMI, BtgZI, EarI, BspQI, SapI,SgeI, BceFI, BslFI, BsoMAI, Bst71I, FaqI, AceIII, BbvII, BveI, LguI,BfuCI, DpnII, FatI, MboI, MluCI, Sau3AI, Tsp509I, BssKI, PspGI, StyD4I,Tsp45I, AoxI, BscFI, Bsp143I, BssMI, BseENII, BstMBI, Kzo9I, NedII,Sse9I, TasI, TspEI, AjnI, BstSCI, EcoRII, MaeIII, NmuCI, Psp6I, MnlI,BspCNI, BsrI, BtsCI, HphI, HpyAV, MboII, AcuI, BciVI, BmrI, BpmI, BpuEI,BseRI, BsgI, BsmI, BsrDI, BtsI, EciI, MmeI, NmeAIII, Hin4II, TscAI,Bce83I, BmuI, BsbI, BscCI, NlaIII, Hpy99I, TspRI, FaeI, Hin1II, Hsp92II,SetI, TaiI, TscI, TscAI, TseFI, Nb.BsrDI, Nb.BtsI, AspCNI, BscGI,BspNCI, EcoHI, FinI, TsuI, UbaF11I, UnbI, Vpak11AI, BspGI, DrdII,Pfl1108I, UbaPI, Nt.AlwI, Nt.BsmAI, Nt.BstNBI, and Nt.BspQI, andvariants thereof. In some embodiments, the first agent comprisesessentially the same function, recognizes the same or essentially thesame recognition sequence, or cuts at the same or essentially samecutting site as any of the listed first agents and variants. In someembodiments, the kit further comprises a second cleaving agent. In someembodiments, the second cleaving agent comprises and enzyme selectedfrom the group consisting of MlyI, SchI, AlwI, BccI, BceAI, BsmAI,BsmFI, FokI, HgaI, PleI, SfaNI, BfuAI, BsaI, BspMI, BtgZI, EarI, BspQI,SapI, SgeI, BceFI, BslFI, BsoMAI, Bst71I, FaqI, AceIII, BbvII, BveI,LguI, BfuCI, DpnII, FatI, MboI, MluCI, Sau3AI, Tsp509I, BssKI, PspGI,StyD4I, Tsp45I, AoxI, BscFI, Bsp143I, BssMI, BseENII, BstMBI, Kzo9I,NedII, Sse9I, TasI, TspEI, AjnI, BstSCI, EcoRII, MaeIII, NmuCI, Psp6I,MnlI, BspCNI, BsrI, BtsCI, HphI, HpyAV, MboII, AcuI, BciVI, BmrI, BpmI,BpuEI, BseRI, BsgI, BsmI, BsrDI, BtsI, EciI, MmeI, NmeAIII, Hin4II,TscAI, Bce83I, BmuI, BsbI, BscCI, NlaIII, Hpy99I, TspRI, FaeI, Hin1II,Hsp92II, SetI, TaiI, TscI, TscAI, TseFI, Nb.BsrDI, Nb.BtsI, AspCNI,BscGI, BspNCI, EcoHI, FinI, TsuI, UbaF11I, UnbI, Vpak11AI, BspGI, DrdII,Pfl1108I, UbaPI, Nt.AlwI, Nt.BsmAI, Nt.BstNBI, and Nt.BspQI, andvariants thereof. In some embodiments, the second agent comprisesessentially the same function, recognizes the same or essentially thesame recognition sequence, or cuts at the same or essentially samecutting site as any of the listed second agents and variants. In someembodiments, the first cleaving agents comprises MlyI. In someembodiments, the second cleaving agent comprises BciVI or BfuCI.

In yet another aspect, the invention relates to a method of nucleic acidamplification, comprising: (a) providing a sample comprising ncircularized single stranded nucleic acids, each comprising a differenttarget sequence; (b) providing a first adaptor that is hybridizable toat least one adaptor hybridization sequence on m of the n circularizedsingle stranded nucleic acids; (c) providing conditions suitable forextending the first adaptor using the m circularized single strandednucleic acids as a template, thereby generating m single strandedamplicon nucleic acids, wherein each of the m single stranded ampliconnucleic acids comprises a plurality of replicas of the target sequencefrom its template; (d) generating double stranded recognition sites fora first agent on the m single stranded amplicon nucleic acids; and (e)providing the first agent under conditions suitable for the first agentto cut the m single stranded amplicon nucleic acids at a plurality ofcutting sites, thereby generating a plurality of single strandedreplicas of the target sequences in the m circularized single strandednucleic acids. In some embodiments, the double stranded recognitionsites comprise a first portion of the first adaptor on a first strand ofthe double stranded recognition sites and a second strand of the firstadaptor on the second strand of the double stranded recognition sites.In some embodiments, the adaptor comprises a palindromic sequence. Insome embodiments, the double stranded recognition sites are generated byhybridizing the first and second portions of the first adaptor to eachother. In some embodiments, the m single stranded amplicon nucleic acidscomprise a plurality of double stranded self-hybridized regions.

In a yet further aspect, the invention relates to a method forgenerating a long nucleic acid molecule, the method comprising the stepsof: (a) providing a plurality of nucleic acids immobilized on a surface,wherein said plurality of nucleic acids comprises nucleic acids havingoverlapping complementary sequences; (b) releasing said plurality ofnucleic acids into solution; and (c) providing conditions promoting: i)hybridization of said overlapping complementary sequences to form aplurality of hybridized nucleic acids; and ii) extension or ligation ofsaid hybridized nucleic acids to synthesize the long nucleic acidmolecule.

In another aspect, the invention relates to an automated system capableof processing one or more substrates, comprising: an inkjet print headfor spraying a microdroplet comprising a chemical species on asubstrate; a scanning transport for scanning the substrate adjacent tothe print head to selectively deposit the microdroplet at specifiedsites; a flow cell for treating the substrate on which the microdropletis deposited by exposing the substrate to one or more selected fluids;an alignment unit for aligning the substrate correctly relative to theprint head each time when the substrate is positioned adjacent to theprint head for deposition; and not comprising a treating transport formoving the substrate between the print head and the flow cell fortreatment in the flow cell, wherein said treating transport and saidscanning transport are different elements.

In yet another aspect, the invention relates to an automated system forsynthesizing oligonucleotides on a substrate, said automated systemcapable of processing one or more substrates, comprising: an inkjetprint head for spraying a solution comprising a nucleoside or activatednucleoside on a substrate; a scanning transport for scanning thesubstrate adjacent to the print head to selectively deposit thenucleoside at specified sites; a flow cell for treating the substrate onwhich the monomer is deposited by exposing the substrate to one or moreselected fluids; an alignment unit for aligning the substrate correctlyrelative to the print head each time when the substrate is positionedadjacent to the print head for deposition; and not comprising a treatingtransport for moving the substrate between the print head and the flowcell for treatment in the flow cell, wherein said treating transport andsaid scanning transport are different elements.

In yet a further aspect, the invention relates to an automated systemcomprising: an inkjet print head for spraying a microdroplet comprisinga chemical species on a substrate; a scanning transport for scanning thesubstrate adjacent to the print head to selectively deposit themicrodroplet at specified sites; a flow cell for treating the substrateon which the microdroplet is deposited by exposing the substrate to oneor more selected fluids; and an alignment unit for aligning thesubstrate correctly relative to the print head each time when thesubstrate is positioned adjacent to the print head for deposition; andwherein the system does NOT comprise a treating transport for moving thesubstrate between the print head and the flow cell for treatment in theflow cell.

With the above in mind, reference is made more specifically to thedrawings which, for illustrative purposes, show the present inventionembodied in compositions, systems and methods in FIGS. 1A-1C and 2A-2C.It will be appreciated that the methods, systems, and compositions mayvary in configuration and in the details of the individual parts invarious embodiments of the invention. Further, the methods may vary indetail and the order of the events or acts. In various embodiments, theinvention is described primarily in terms of use with nucleic acids, inparticular, DNA oligomers and polynucleotides. It should be understood,however, that the invention may be used with a variety of differenttypes of molecules, including RNA or other nucleic acids, peptides,proteins, or other molecules of interest. Suitable building blocks foreach of these larger molecules of interest are known in the art.

The present invention provides compositions, systems, and methods usefulin the preparation and the synthesis of libraries of molecules ofinterest, including nucleic acids, polypeptides, proteins andcombinations thereof. In various embodiments, the invention contemplatesthe use of static and dynamic wafers, e.g. those that are manufacturedfrom silicon substrates, for performing micro-, nano-, or picoliterscale reactions in parallel. In addition, the same can be applied tomicro-, nano-, or picoliter manipulation of fluids in parallel to allowfor linking a plurality of reactions in resolved volumes. Themanipulation of fluids may comprise flowing, combining, mixing,fractionation, generation of drops, heating, condensation, evaporation,sealing, stratification, pressurizing, drying, or any other suitablefluid manipulation known in the art. In various embodiments, the wafersprovide architectures for fluid manipulation that are built into thesurface. Features of varying shape and size may be architected inside orthrough a wafer substrate. The methods and compositions of theinvention, in various embodiments, make use of specifically architecteddevices exemplified in further detail herein, for the synthesis ofbiological molecules. In particular, the invention provides for the denovo synthesis of large, high-density libraries comprising long,high-quality oligonucleotides and polynucleotides, e.g. using standardphosphoramidite chemistry and suitable gene assembly techniques, byprecisely controlling reaction conditions such as time, dosage andtemperature.

Referring now to FIG. 1C, the invention in various embodimentscontemplates the use of one or more static or dynamic wafers for fluidmanipulation. The wafers may be constructed from a number of suitablematerials as further described herein, e.g. silicon. Nanoreactor wafersmay be configured to receive and hold liquids in a plurality offeatures. Additional wafers, for example those that are used for in situsynthesis reactions, maybe contacted with nanoreactor wafers to collectand/or mix liquids. The nanoreactors may collect liquids from aplurality of additional wafers. Typically, nanoreactors are aligned withone or more resolved loci on additional wafers when the nanoreactorwafer is contacted. Reagents and solvents may be provided within thenanoreactor prior to contact. Alternatively, nanoreactors may be emptyprior to contacting an additional wafer. In some embodiments,nanoreactors collect oligonucleotides synthesized in one or moreresolved locus of a DNA synthesis wafer. These oligonucleotides can beassembled into a longer gene within the nanoreactor. The nanoreactorsmay be sealed upon alignment and contact of an additional wafer by anysuitable means, e.g. capillary burst valves, pressure, adhesives, or anyother suitable sealing means known the art. The seal may be releasable.Reactions within the nanoreactor wafer may be carried out in sealedvolumes and may comprise temperature cycling, e.g. as applied in PCR orPCA. Isothermal reactions, such as isothermal amplification, are furtherwithin the bounds of the invention. The DNA synthesis wafers may beconfigured to perform in situ synthesis of oligonucleotides at resolvedloci on or inside the surface with precise control. An inkjet printheadmay be utilized to deliver drops of reagents for synthesis, e.g.standard phosphoramidite synthesis onto the resolved loci of thesynthesis wafer. Other reagents that are common to a plurality ofresolved loci may be passed through them in bulk. In some embodiments,DNA synthesis wafers are replaced with synthesis wafers for the in situsynthesis of molecules other than DNA oligonucleotides as furtherdescribed elsewhere herein. Thus, the invention contemplates fastsynthesis of large libraries of oligonucleotides and long genes withhigh-quality through the precise control of reaction conditions in aplurality of small volumes. A further benefit of the invention is areduced reagent use in comparison to the traditional synthesis methodsknown in the art.

Various methods are contemplated for the de novo synthesis of genelibraries with low error rates. FIGS. 2A-2C illustrates exemplaryapplications of the methods and compositions of the invention for thesynthesis of large, high quality gene libraries with long sequences inparallel. In various embodiments, static and dynamic wafers enable aplurality of reactions in a process flow. For example, oligonucleotidesynthesis typically in situ on a DNA synthesis wafer, may be followed bya gene assembly reaction, such as polymerase cycling assembly (PCA), ofthe synthesized oligonucleotides into longer sequences. The assembledsequences may be amplified, e.g. through PCR. Suitable error correctionreactions described herein or known in the art can be used to minimizethe number of assembled sequences that deviate from a target sequence.Sequencing libraries may be built and a fraction of the product may bealiquoted for sequencing, such as next generation sequencing (NGS).

The gene synthesis processes as exemplified in FIGS. 2A-2C may beadjusted according to the needs of a requester. According to the resultsobtained from an initial sequencing step, e.g. NGS, the assembled geneswith acceptable error rates may be shipped, e.g. on a plate, to arequester (FIG. 2B). The methods and compositions of the invention allowfor error rates less than about 1/10 kb to be easily achieved, althoughalternative error thresholds may be set as described in further detailelsewhere herein. To achieve higher degrees of purity, de novosynthesized/assembled sequences may be cloned purified from singlecolonies. The identity of a correct desired sequence may be testedthrough sequencing, e.g. NGS. Optionally, a higher confidence for theaccuracy of the sequencing information may be obtained, e.g. via anothersequencing method such as Sanger sequencing. Verified sequences may beshipped, e.g. on a plate, to a requester (FIG. 2C) Methods forgeneration of sequencing libraries are described in further detailelsewhere herein.

Substrates/Wafers

In an aspect, a substrate having a functionalized surface made by any ofthe methods described herein and methods of synthesizingoligonucleotides on the substrate having a functionalized surface aredescribed herein. The substrate can comprise a solid support having aplurality of resolved loci. The plurality of resolved loci may have anygeometry, orientation or organization. The resolved loci may be in anyscale (e.g., micro-scale or nano-scale), or contain microstructuresfabricated into the substrate surface. The resolved loci can belocalized on microchannels with at least one dimension. Individualresolved loci of a substrate may be fluidically disconnected from eachother, e.g. a first resolved locus for the synthesis of a firstoligonucleotide may be on a first via between the two surfaces of asubstrate and a second resolved locus for the synthesis of a secondoligonucleotide may be on a second via between the two surfaces of asubstrate, the first and second vias not being fluidically connectedwithin the substrate, but starting and ending from the same two surfacesof the substrate. In some cases, the microstructure of resolved loci canbe microchannels or microwells in 2-D or 3-D. A “3-D” microchannel meansthe cavity of the microchannel can be interconnected or extend withinthe solid support. Within the microchannels or microwells, there can besecondary microstructures or features with any geometry, orientation ororganization. The surface of the secondary features may befunctionalized with a moiety that can decrease the surface energy of thesurface of the secondary features. Droplets of reagents for synthesizingoligonucleotides can be deposited into the microchannels or microwells.A microwell, as used herein, refers to a structure of microfluidic scalethat can hold a liquid. In various embodiments, microwells allow liquidflow between a top and a bottom end, through a fluidic opening on eachend, therefore acting like a microchannel. In these contexts, the termsmicrowell and microchannel are used interchangeably throughout thespecification.

FIG. 3 illustrates an example of the system for oligonucleotidesynthesis comprising a first substrate and, optionally, a secondsubstrate as described herein. The inkjet printer printheads can move inX-Y direction to the location of the first substrate. A second substratecan move in Z direction to seal with the first substrate, forming aresolved reactor. The synthesized oligonucleotides can be delivered fromthe first substrate to the second substrate. In another aspect, currentinvention also concerns a system for oligonucleotide assembly. Thesystem for oligonucleotide assembly can comprise a system for waferhandling. FIG. 4 illustrates an example for the layout design of asubstrate, according to various embodiments of the invention. Thesubstrate can comprise a plurality of microwells and the microwells canbe arrayed on a uniform pitch, e.g. a 1.5 mm pitch. Alternatively,multiple pitches may be picked in different directions of the layout,for example, rows of microstructures can be defined by a first pitch andwithin each row, the microstructures may be separated by a second pitch.The pitch may comprise any suitable size, e.g. 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, or5 mm. The microwell can be designed having any suitable dimensions, forexample a diameter of 80 μm as exemplified in FIG. 4, or any suitablediameter, including 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 μm, and themicrowells can be connected to a plurality of smaller microwells. Thesurface of the smaller microwells can be functionalized at selectedregions facilitating liquid of reagents to flow into, for example via ahigh energy surface functionalization. As illustrated in FIG. 4, thediameter of the smaller microwells can be about 20 μm, or any suitablediameter, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 65, 70, 75 or 80 μm. FIG. 5 illustrates a case when adroplet of reagent is deposited into a microwell by an inkjet printer.The liquid droplet can spread over and fill the smaller microwells, insome cases facilitated by a high energy surface modification of thesurface of the microwells in comparison adjacent surfaces.

Having a high density of resolved loci on the substrate having afunctionalized surface may be desirable for having a small device and/orsynthesizing a large number of molecules with a small device and/orsynthesizing a large number of different molecules. The functionalizedsurface of the substrate may comprise any suitable density of resolvedloci (e.g., a density suitable for synthesizing oligonucleotides with agiven number of total different oligonucleotides to be synthesized,given amount of time for the synthesis process, or for a given cost peroligonucleotide, gene, or library). In some embodiments, the surface hasa density of resolved loci of about 1, about 2, about 3, about 4, about5, about 6, about 7, about 8, about 9, about 10, about 15, about 20,about 25, about 30, about 35, about 40, about 50, about 75, about 100,about 200, about 300, about 400, about 500, about 600, about 700, about800, about 900, about 1000, about 1500, about 2000, about 3000, about4000, about 5000, about 6000, about 7000, about 8000, about 9000, about10000, about 20000, about 40000, about 60000, about 80000, about 100000,or about 500000 sites per 1 mm². In some embodiments, the surface has adensity of resolved loci of at least about 50, at least 75, at leastabout 100, at least about 200, at least about 300, at least about 400,at least about 500, at least about 600, at least about 700, at leastabout 800, at least about 900, at least about 1000, at least about 1500,at least about 2000, at least about 3000, at least about 4000, at leastabout 5000, at least about 6000, at least about 7000, at least about8000, at least about 9000, at least about 10000, at least about 20000,at least about 40000, at least about 60000, at least about 80000, atleast about 100000, or at least about 500000 sites per 1 mm². Theresolved loci on the substrate can have any different organization. Forexample without limitations, the resolved loci can be clustered in closeproximity to form one or more circular region, rectangular region,elliptical region, irregular region and the like. In an aspect, theresolved loci are closely packed and have a low amount or no amount ofcross-contamination (e.g., the droplets of reagents that are depositedinto one resolved locus will not substantially mix with the droplets ofreagents that are deposited into another nearest resolved locus). Theorganization of the resolved loci on the substrate can be designed suchthat it allows each sub-region or the entire region to be coveredtogether creating a sealed cavity with controlled humidity, pressure orgas content in the sealed cavity so that the each sub-region or theentire region can have the same humidity, pressure or gas content, orsubstantially similar humidity, pressure or gas content as allowed underfluidically connected conditions. Some examples of different designs forthe resolved loci on the substrate are illustrated in FIG. 6. Forexample, FIG. 6B part b is a design of a layout referred to as Array ofHoles; FIG. 6B part c is a design of a layout referred to as Flowers;FIG. 6B part d is a design of a layout referred to as Gunsight; and FIG.6B part e is a design of a layout referred to as Radial Flower. FIG. 6Cexemplifies a design of the substrate covered with a series ofmicrowells on a 97.765 μm stencil. The microwells as exemplified in FIG.6C are clustered into islands. The microwells can be filled withreagents from the inkjet head.

Each of the resolved loci on the substrate can have any shape that isknown in the art, or the shapes that can be made by methods known in theart. For example, each of the resolved loci can have an area that is ina circular shape, a rectangular shape, elliptical shape, or irregularshape. In some embodiments, the resolved loci can be in a shape thatallows liquid to easily flow through without creating air bubbles. Insome embodiments, the resolved loci can have a circular shape, with adiameter that can be about, at least about, or less than about 1micrometers (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm,11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm,150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 250 μm, 300 μm, 350 μm,400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm or 750 μm. Theresolved loci may have a monodisperse size distribution, i.e. all of themicrostructures may have approximately the same width, height, and/orlength. Alternatively, the resolved loci of may have a limited number ofshapes and/or sizes, for example the resolved loci may be represented in2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or more distinct shapes, eachhaving a monodisperse size. In some embodiments, the same shape can berepeated in multiple monodisperse size distributions, for example, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or more monodisperse sizedistributions. A monodisperse distribution may be reflected in aunimodular distribution with a standard deviation of less than 25%, 20%,15%, 10%, 5%, 3%, 2%, 1%, 0.1%, 0.05%, 0.01%, 0.001% of the mode orsmaller.

A substrate having a high density of resolved loci typically results ina resolved locus within a small area. Consequently, it can result in asmall microchannel. The microchannels can contain deposited droplets ofreagents in different volumes. The microchannels can have any suitabledimensions that allow sufficiently large surface areas and/or volumesfor the various embodiments of the invention. In an aspect, the volumeof the microchannel is suitably large such that a reagent in a dropletthat is deposited in the microchannel is not fully depleted during theoligonucleotide synthesis. In these aspects, amongst others, the volumeof a well structure can guide the time period or density with whicholigonucleotides can be synthesized.

Each of the resolved loci can have any suitable area for carrying outthe reactions according to various embodiments of the inventiondescribed herein. In some cases, the plurality of resolved loci canoccupy any suitable percentage of the total surface area of thesubstrate. In some cases, the area of the resolved loci can be thecross-sectional area of microchannels or microwells built into asubstrate. In some embodiments, the plurality of the microstructures orresolved loci directly can occupy about, at least about, or less thanabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% of the surface of the substrate. In someembodiments, the plurality of resolved loci can occupy about, at leastabout, or less than about 10 mm², 11 mm², 12 mm², 13 mm², 14 mm², 15mm², 16 mm², 17 mm², 18 mm², 19 mm², 20 mm², 25 mm², 30 mm², 35 mm², 40mm², 50 mm², 75 mm², 100 mm², 200 mm², 300 mm², 400 mm², 500 mm², 600mm², 700 mm², 800 mm², 900 mm², 1000 mm², 1500 mm², 2000 mm², 3000 mm²,4000 mm², 5000 mm², 7500 mm², 10000 mm², 15000 mm², 20000 mm², 25000mm², 30000 mm², 35000 mm², 40000 mm², 50000 mm², 60000 mm², 70000 mm²,80000 mm², 90000 mm², 100000 mm², 200000 mm², 300000 mm², or more oftotal area.

The microstructures built into a substrate may comprise microchannels ormicrowells, wherein the microstructures start from a top or bottomsurface of the substrate and in some cases are fluidically connected toa typically opposing surface (e.g. bottom or top). The terms “top” and“bottom” do not necessarily relate to the position of the substrate withrespect to gravity at any given time, but are generally used forconvenience and clarity. The microchannels or microwells can have anysuitable depth or length. In some cases, the depth or length of themicrochannel or microwell is measured from the surface of the substrate(and/or bottom of the solid support) to the top of the solid support. Insome cases, the depth or length of the microchannel or microwell isapproximately equal to the thickness of the solid support. In someembodiments, the microchannels or microwells are about, less than about,or greater than about 1 micrometer (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7μm, 8 μm, 9 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm,50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm,100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 300 μm, 400 μm or 500 μm deep orlong. The microchannels or microwells can have any length of perimeterthat is suitable for the embodiments of the invention described herein.In some cases, the perimeter of the microchannel or microwell ismeasured as the perimeter of a cross-sectional area, e.g. a crosssectional area that is perpendicular to fluid flow direction throughsaid microchannel or microwell. In some embodiments, the microchannelsor microwells have about, less than about, or at least about 1micrometer (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm,15 μm, 20 μm, 25 μm, 30 μm, 31 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 125 μm, 150μm, 175 μm, 200 μm, 300 μm, 400 μm or 500 μm in perimeter. In someembodiments, the nominal arclength density of the microchannels ormicrowells can have any suitable arclength per μm² of the planarsubstrate area. As described herein, the arclength density refers to thelength of the perimeters of the cross-sections of the microchannels ormicrowells per surface area of the planar substrate. For example,without limitation, the nominal arclength density of the microchannelsor microwells can be at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.006,0.007, 0.008, 0.009, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045,0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 μm/μm², or more. In someembodiments, the nominal arclength density of the microchannels ormicrowells can be 0.036 μm/μm². In some embodiments, the nominalarclength density of the microchannels or microwells can be at least0.001 μm/μm². In some embodiments, the nominal arclength density of themicrochannels or microwells can be at least 0.01 μm/μm². Further, thenominal surface area of the microchannels or microwells that is suitablefor reactions described herein, e.g. through surface coating with asuitable moiety, can be maximized. The surface area of the microchannelsor microwells that is coated with suitable moieties as described hereincan facilitate the attachment of oligonucleotides to the surface. Insome embodiments, the nominal surface area of the microchannels ormicrowells suitable for reactions described herein, such asoligonucleotide synthesis, is at least 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45,1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5 or 5 μm² of the planarsubstrate area.

The microchannels or microwells can have any volume that is suitable forthe methods and compositions described herein. In some embodiments, themicrochannels or microwells have a volume that is less than about 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 picoliter (pl), lessthan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950 or 990 nanoliter (nl), less than about 0.5 microliters (μl), lessthan about 1 μl, less than about 1.5 μl, less than about 2 μl, less thanabout 2.5 μl, less than about 3 μl, less than about 3.5 μl, less thanabout 4 μl, less than about 4.5 μl, less than about 5 μl, less thanabout 5.5 μl, less than about 6 μl, less than about 6.5 μl, less thanabout 7 μl, less than about 7.5 μl, less than about 8 μl, less thanabout 8.5 μl, less than about 9 μl, less than about 9.5 μl, less thanabout 10 μl, less than about 11 μl, less than about 12 μl, less thanabout 13 μl, less than about 14 μl, less than about 15 μl, less thanabout 16 μl, less than about 17 μl, less than about 18 μl, less thanabout 19 μl, less than about 20 μl, less than about 25 μl, less thanabout 30 μl, less than about 35 μl, less than about 40 μl, less thanabout 45 μl, less than about 50 μl, less than about 55 μl, less thanabout 60 μl, less than about 65 μl, less than about 70 μl, less thanabout 75 μl, less than about 80 μl, less than about 85 μl, less thanabout 90 μl, less than about 95 μl or less than about 100 μl. In someembodiments, the microchannels or microwells have a volume that is equalto or greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900 or 950 picoliter (pl), equal or greater than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950 or 990 nanoliter (nl), equalor greater than about 0.5 microliters (μl), about 1 μl, about 1.5 μl,about 2 μl, about 2.5 μl, about 3 μl, about 3.5 μl, about 4 μl, about4.5 μl, about 5 μl, about 5.5 μl, about 6 μl, about 6.5 μl, about 7 μl,about 7.5 μl, about 8 about 8.5 μl, about 9 μl, about 9.5 μl, about 10μl, about 11 μl, about 12 μl, about 13 μl, about 14 μl, about 15 μl,about 16 μl, about 17 μl, about 18 μl, about 19 μl, about 20 μl, about25 μl, about 30 μl, about 35 μl, about 40 μl, about 45 μl, about 50 μl,about 55 μl, about 60 μl, about 65 μl, about 70 μl, about 75 μl, about80 μl, about 85 μl, about 90 μl, about 95 μl or about 100 μl.

The microchannels or microwells can have an aspect ratio of less than 1.As used herein, the term “aspect ratio,” refers to the ratio of achannel's width to that channel's depth. Thus, a channel having anaspect ratio of less than 1, is deeper than it is wide, while a channelhaving an aspect ratio greater than 1 is wider than it is deep. In someaspects, the aspect ratio of the microchannels or microwells can be lessthan or equal to about 0.5, about 0.2, about 0.1, about 0.05 or less. Insome embodiments, the aspect ratio of the microchannels or microwellscan be about 0.1. In some embodiments, the aspect ratio of themicrochannels or channels can be about 0.05. The microstructuresdescribed herein, e.g., microchannels or microwells having aspect ratiosless than 1, 0.1 or 0.05, may include channels having one, two, three,four, five, six or more corners, turns, and the like. Themicrostructures described herein may include the aspect ratiosdescribed, e.g., less than 1, 0.1 or 0.05, with respect to allmicrochannels or microwells contained within a particular resolvedlocus, e.g., one or more intersecting channels, some of these channels,a single channel and even a portion or portions of one or moremicrochannels or microwells. Other designs and methods of fabricatingthe microchannels with low aspect ratios are described in U.S. Pat. No.5,842,787, which is incorporated herein by reference.

The microstructures such as microchannels or microwells on a substratehaving a plurality of resolved loci can be manufactured by any methodthat is described herein or otherwise known in the art (e.g.,microfabrication processes). Microfabrication processes that may be usedin making the substrate disclosed herein include without limitationlithography; etching techniques such as wet chemical, dry, andphotoresist removal; microelectromechanical (MEMS) techniques includingmicrofluidics/lab-on-a-chip, optical MEMS (also called MOEMS), RF MEMS,PowerMEMS, and BioMEMS techniques and deep reactive ion etching (DRIE);nanoelectromechanical (NEMS) techniques; thermal oxidation of silicon;electroplating and electroless plating; diffusion processes such asboron, phosphorus, arsenic, and antimony diffusion; ion implantation;film deposition such as evaporation (filament, electron beam, flash, andshadowing and step coverage), sputtering, chemical vapor deposition(CVD), epitaxy (vapor phase, liquid phase, and molecular beam),electroplating, screen printing, and lamination. See generally Jaeger,Introduction to Microelectronic Fabrication (Addison-Wesley PublishingCo., Reading Mass. 1988); Runyan, et al., Semiconductor IntegratedCircuit Processing Technology (Addison-Wesley Publishing Co., ReadingMass. 1990); Proceedings of the IEEE Micro Electro Mechanical SystemsConference 1987-1998; Rai-Choudhury, ed., Handbook of Microlithography,Micromachining & Microfabrication (SPIE Optical Engineering Press,Bellingham, Wash. 1997).

In an aspect, a substrate having a plurality of resolved loci can bemanufactured using any method known in the art. In some embodiments, thematerial of the substrate having a plurality of resolved loci can be asemiconductor substrate such as silicon dioxide. The materials of thesubstrate can also be other compound III-V or II-VI materials, such asGallium arsenide (GaAs), a semiconductor produced via the Czochralskiprocess (Grovenor, C. (1989). Microelectronic Materials. CRC Press. pp.113-123). The material can present a hard, planar surface that exhibitsa uniform covering of reactive oxide (—OH) groups to a solution incontact with its surface. These oxide groups can be the attachmentpoints for subsequent silanization processes. Alternatively, alipophillic and hydrophobic surface material can be deposited thatmimics the etching characteristics of silicon oxide. Silicon nitride andsilicon carbide surfaces may also be utilized for the manufacturing ofsuitable substrates according to the various embodiments of theinvention.

In some embodiments, a passivation layer can be deposited on thesubstrate, which may or may not have reactive oxide groups. Thepassivation layer can comprise silicon nitride (Si₃N₄) or polymide. Insome instances, a photolithographic step can be used to define regionswhere the resolved loci form on the passivation layer.

The method for producing a substrate having a plurality of resolved locican start with a substrate. The substrate (e.g., silicon) can have anynumber of layers disposed upon it, including but not limited to aconducting layer such as a metal. The conducting layer can be aluminumin some instances. In some cases, the substrate can have a protectivelayer (e.g., titanium nitride). In some cases, the substrate can have achemical layer with a high surface energy. The layers can be depositedwith the aid of various deposition techniques, such as, for example,chemical vapor deposition (CVD), atomic layer deposition (ALD), plasmaenhanced CVD (PECVD), plasma enhanced ALD (PEALD), metal organic CVD(MOCVD), hot wire CVD (HWCVD), initiated CVD (iCVD), modified CVD(MCVD), vapor axial deposition (VAD), outside vapor deposition (OVD) andphysical vapor deposition (e.g., sputter deposition, evaporativedeposition).

In some cases, an oxide layer is deposited on the substrate. In someinstances, the oxide layer can comprise silicon dioxide. The silicondioxide can be deposited using tetraethyl orthosilicate (TEOS), highdensity plasma (HDP), or any combination thereof.

In some instances, the silicon dioxide can be deposited using a lowtemperature technique. In some cases, the process is low-temperaturechemical vapor deposition of silicon oxide. The temperature is generallysufficiently low such that pre-existing metal on the chip is notdamaged. The deposition temperature can be about 50° C., about 100° C.,about 150° C., about 200° C., about 250° C., about 300° C., about 350°C., and the like. In some embodiments, the deposition temperature isbelow about 50° C., below about 100° C., below about 150° C., belowabout 200° C., below about 250° C., below about 300° C., below about350° C., and the like. The deposition can be performed at any suitablepressure. In some instances, the deposition process uses RF plasmaenergy.

In some cases, the oxide is deposited by a dry thermally grown oxideprocedure (e.g., those that may use temperatures near or exceeding1,000° C.). In some cases, the silicon oxide is produced by a wet steamprocess.

The silicon dioxide can be deposited to a thickness suitable for themanufacturing of suitable microstructures described in further detailelsewhere herein.

The silicon dioxide can be deposited to any suitable thickness. In someembodiments, the silicon dioxide layer may have a thickness of at leastor at least about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm,10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 125 nm, 150nm, 175 nm, 200 nm, 300 nm, 400 nm or 500 nm, 1 μm, 1.1 μm, 1.2 μm, 1.3μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, or more. Thesilicon dioxide layer may have a thickness of at most or at most about2.0 μm, 1.9 μm, 1.8 μm, 1.7 μm, 1.6 μm, 1.5 μm, 1.4 μm, 1.3 μm, 1.2 μm,1.1 μm, 1.0 μm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm,100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm,50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 9 nm, 8,nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, or less. The silicondiooxide layer may have a thickness that is between 1.0 nm-2.0 μm,1.1-1.9 μm, 1.2-1.8 nm, 1.3-1.7 μm, 1.4-1.6 μm. Those of skills in theart will appreciate that The silicon diooxide layer may have a thicknessthat falls within any range bound by any of these values, for example(1.5-1.9 μm). The silicon dioxide may have a thickness that falls withinany range defined by any of the values serving as endpoints of therange. The resolved loci (e.g., microchannels or microwells) can becreated in a silicon dioxide substrate using various manufacturingtechniques that are known in the art. Such techniques may includesemiconductor fabrication techniques. In some cases, the resolved lociare created using photolithographic techniques such as those used in thesemiconductor industry. For example, a photo-resist (e.g., a materialthat changes properties when exposed to electromagnetic radiation) canbe coated onto the silicon dioxide (e.g., by spin coating of a wafer) toany suitable thickness. The substrate including the photo-resist can beexposed to an electromagnetic radiation source. A mask can be used toshield radiation from portions of the photo-resist in order to definethe area of the resolved loci. The photo-resist can be a negative resistor a positive resist (e.g., the area of the resolved loci can be exposedto electromagnetic radiation or the areas other than the resolved locic_(a)n _(b)e exposed to electromagnetic radiation as defined by themask). The area overlying the location in which the resolved loci are tobe created is exposed to electromagnetic radiation to define a patternthat corresponds to the location and distribution of the resolved lociin the silicon dioxide layer. The photoresist can be exposed toelectromagnetic radiation through a mask defining a pattern thatcorresponds to the resolved loci. Next, the exposed portion of thephotoresist can be removed, such as, e.g., with the aid of a washingoperation (e.g., deionized water). The removed portion of the mask canthen be exposed to a chemical etchant to etch the substrate and transferthe pattern of resolved loci into the silicon dioxide layer. The etchantcan include an acid, such as, for example, sulfuric acid (H₂SO₄). Thesilicon dioxide layer can be etched in an anisotropic fashion. Using themethods described herein, high anisotropy manufacturing methods, such asDRIE can be applied to fabricate microstructures, such as microwells ormicrochannels comprising loci of synthesis, on or within a substratewith side walls that deviate less than about ±3°, 2°, 1°, 0.5°, 0.1°, orless from the vertical with respect to the surface of the substrate.Undercut values of less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5,0.1 μm or less can be achieved resulting in highly uniformmicrostructures.

Various etching procedures can be used to etch the silicon dioxide inthe area where the resolved loci are to be formed. The etch can be anisotropic etch (i.e., the etch rate alone one direction substantiallyequal or equal to the etch rate along an orthogonal direction), or ananisotropic etch (i.e., the etch rate along one direction is less thanthe etch rate alone an orthogonal direction), or variants thereof. Theetching techniques can be both wet silicon etches such as KOH, TMAH, EDPand the like, and dry plasma etches (for example DRIE). Both may be usedto etch micro structures wafer through interconnections.

In some cases, an anisotropic etch removes the majority of the volume ofthe resolved loci. Any suitable percentage of the volume of the resolvedloci can be removed including about 60%, about 70%, about 80%, about90%, or about 95%. In some cases, at least about 60%, at least about70%, at least about 80%, at least about 90%, or at least about 95% ofthe material is removed in an anisotropic etch. In some cases, at mostabout 60%, at most about 70%, at most about 80%, at most about 90%, orat most about 95% of the material is removed in an anisotropic etch. Insome embodiments, the anisotropic etch does not remove silicon dioxidematerial all of the way through the substrate. An isotropic etch is usedto remove material all of the way through the substrate creating a hole,according to some embodiments.

In some cases, the wells are etched using a photo-lithographic step todefine the resolved loci followed by a hybrid dry-wet etch. Thephoto-lithographic step can comprise coating the silicon dioxide with aphoto-resist and exposing the photo-resist to electromagnetic radiationthrough a mask (or reticle) having a pattern that defines the resolvedloci. In some instances, the hybrid dry-wet etch comprises: (a) dryetching to remove the bulk of the silicon dioxide in the regions of theresolved loci defined in the photoresist by the photo-lithographic step;(b) cleaning the substrate; and (c) wet etching to remove the remainingsilicon dioxide from the substrate in the regions of the resolved loci.

The substrate can be cleaned with the aid of a plasma etching chemistry,or exposure to an oxidizing agent, such as, for example, H₂O₂, O₂, O₃,H₂SO₄, or a combination thereof, such as a combination of H₂O₂ andH₂SO₄. The cleaning can comprise removing residual polymer, removingmaterial that can block the wet etch, or a combination thereof. In someinstances, the cleaning is plasma cleaning. The cleaning step canproceed for any suitable period of time (e.g., 15 to 20 seconds). In anexample, the cleaning can be performed for 20 seconds with an AppliedMaterials eMAx-CT machine with settings of 100 mT, 200 W, 20 G, 20 O₂.

The dry etch can be an anisotropic etch that etches substantiallyvertically (e.g., toward the substrate) but not laterally orsubstantially laterally (e.g., parallel to the substrate). In someinstances, the dry etch comprises etching with a fluorine based etchantsuch as CF₄, CHF₃, C₂F₆, C₃F₆, or any combination thereof. In oneinstance, the etching is performed for 400 seconds with an AppliedMaterials eMax-CT machine having settings of 100 mT, 1000 W, 20 G, and50 CF4. The substrates described herein can be etched by deepreactive-ion etching (DRIE). DRIE is a highly anisotropic etch processused to create deep penetration, steep-sided holes and trenches inwafers/substrates, typically with high aspect ratios. The substrates canbe etched using two main technologies for high-rate DRIE: cryogenic andBosch. Methods of applying DRIE are described in the U.S. Pat. No.5,501,893, which is herein incorporated by reference in its entirety.

The wet etch can be an isotropic etch that removes material in alldirections. In some instances, the wet etch undercuts the photo-resist.Undercutting the photo-resist can make the photo-resist easier to removein a later step (e.g., photo-resist “lift off”). In an embodiment, thewet etch is buffered oxide etch (BOE). In some cases, the wet oxideetches are performed at room temperature with a hydrofluoric acid basethat can be buffered (e.g., with ammonium fluoride) to slow down theetch rate. Etch rate can be dependent on the film being etched andspecific concentrations of HF and/or NH₄F. The etch time needed tocompletely remove an oxide layer is typically determined empirically. Inone example, the etch is performed at 22° C. with 15:1 BOE (bufferedoxide etch).

The silicon dioxide layer can be etched up to an underlying materiallayer. For example, the silicon dioxide layer can be etched until atitanium nitride layer.

In an aspect, a method for preparing a substrate having a plurality ofresolved loci comprises etching the resolved loci such as microwells ormicrochannels into a substrate, such as a silicon substrate comprising asilicon dioxide layer coated thereon using (a) a photo-lithographic stepto define the resolved loci; (b) a dry etch to remove the bulk of thesilicon dioxide in the regions of the resolved loci defined by thephoto-lithographic step; and (c) a wet etch to remove the remainingsilicon dioxide from the substrate in the regions of the resolved loci.In some cases, the method further comprises removing residual polymer,removing material that can block the wet etch, or a combination thereof.The method can include a plasma cleaning step.

In some embodiments, the photo-resist is not removed from the silicondioxide following the photo-lithographic step or the hybrid wet-dry etchin some cases. Leaving the photo-resist can be used to direct metalselectively into the resolved loci and not onto the upper surface of thesilicon dioxide layer in later steps. In some cases, the substrate iscoated with a metal (e.g., aluminum) and the wet etch does not removecertain components on the metal, e.g. those that protect the metal fromcorrosion (e.g., titanium nitride (TiN)). In some cases, however, thephotoresist layer can be removed, such as with the aid of chemicalmechanical planarization (CMP).

Differential Functionalization of Substrates

As described herein, functionalization of a surface, for example thesurface of a silicon wafer, may refer to any process by which thesurface properties of a material are modified by the deposition of achemical species on the surface. A common method for achievingfunctionalization is deposition of an organosilane molecule by chemicalvapor deposition. It can also be done in a wet silanization process.

Differential functionalization, also commonly referred to as “selectivearea deposition” or “selective area functionalization,” may refer to anyprocess that produces two or more distinct areas on a monolithicstructure where at least one area has different surface or chemicalproperties than other areas on the same structure. The propertiesinclude but are not limited to surface energy, chemical termination,surface concentration of a chemical moiety, etc. The different areas maybe contiguous.

Active functionalization may refer to the functionalization of surfacesthat will take part in some downstream production step such as DNAsynthesis, or DNA or protein binding. Thus, a suitable functionalizationmethod as described elsewhere herein or otherwise known in the art, isselected to allow for the particular downstream production step to takeplace on the surface.

Passive functionalization may refer to the functionalization of surfacesthat will render those areas ineffective at the principle function ofthe active areas. For example, if the active functionalization isdesigned to bind DNA, the passive functionalized areas will not bindDNA.

Photoresist typically refers to a light-sensitive material commonly usedin standard industrial processes, such as photolithography, to formpatterned coatings. It is applied as a liquid, but it solidifies on thesubstrate as volatile solvents in the mixture evaporate. It may beapplied in a spin coating process as a thin film (1 um to 100 um) to aplanar substrate. It may be patterned by exposing it to light through amask or reticle, changing its dissolution rate in a developer. It may be“positive” (light exposure increases dissolution) or “negative” (lightexposure decreases dissolution). It may be used as a sacrificial layerthat serves as a blocking layer for subsequent steps that modify theunderlying substrate (such as etching). Once that modification iscomplete, the resist is removed.

Photolithography may refer to a process for patterning substrates. Acommon basic process comprises 1) applying a photoresist to a substrate,2) exposing the resist to light through a binary mask that is opaque insome areas and clear in other areas, and then 3) developing the resistwhich results in patterning the resist based on what areas were exposed.After development, the patterned resist serves as a mask for subsequentprocessing steps, such as etching, ion implantation, or deposition.After the processing steps, the resist is typically removed, for examplevia plasma stripping or wet chemical removal.

In various embodiments, methods using photoresist are employed whereinphotoresist facilitates manufacturing of substrates with differentialfunctionalization.

A series of manufacturing steps may form the baseline of a differentialfunctionalization process, wherein the individual steps may be modified,removed, or supplemented with additional steps to achieve the desiredfunctionalization pattern on a surface, according to the variousembodiments of the invention. First, an initial preparation of thetarget surface may be achieved, for example, by a chemical clean and mayinclude an initial active or passive surface functionalization.

Second, the application of photoresist may be achieved by a variety ofdifferent techniques. In various embodiments, the flow of resist intodifferent parts of the structure is controlled by the design of thestructure, for example by taking advantage of the intrinsic pinningproperties of fluids at various points of the structure, such as atsharp step edges. The photoresist leaves behind a solid film once thetransporting solvents of the resist evaporate.

Third, photolithography may be optionally used to remove the resist incertain specific regions of the substrate so that those regions can befurther modified.

Fourth, plasma descum, a, typically, short plasma cleaning step using,for example, an oxygen plasma, may be used to facilitate the removal ofany residual organic contaminants in the resist cleared areas.

Fifth, the surface may be functionalized while the areas covered inresist are protected from any active or passive functionalization. Anysuitable process that changes the chemical properties of the surfacedescribed herein or known in the art may be used to functionalize thesurface, for example chemical vapor deposition of an organosilane.Typically, this results in the deposition of a self-assembled monolayer(SAM) of the functionalization species.

Sixth, the resist may be stripped and removed, for example by dissolvingit in suitable organic solvents, plasma etching, exposure anddevelopment, etc., thereby exposing the areas of the substrate that hadbeen covered by the resist. In some embodiments, a method that will notremove functionalization groups or otherwise damage the functionalizedsurfaces is selected for the resist strip.

Seventh, a second functionalization step involving active or passivefunctionalization may optionally be performed. In some embodiments, theareas functionalized by the first functionalization step block thedeposition of the functional groups used in the second functionalizationstep.

In various embodiments, differential functionalization facilitatesspatial control of the regions on the chip where DNA is synthesized. Insome embodiments, differential functionalization provides improvedflexibility to control the fluidic properties of the chip. In someembodiments, the process by which oligos are transferred from aoligonucleotide synthesis device to a nanowell device is thereforeimproved by differential functionalization. In some embodiments,differential functionalization provides for the manufacturing ofdevices, for example nanoreactor or oligonucleotide syntheses devices,where the walls of wells or channels are relatively hydrophilic, asdescribed elsewhere herein, and the external surfaces are relativelyhydrophobic, as described elsewhere herein.

FIG. 36 parts A-F illustrates exemplary applications of differentialfunctionalization on the microfluidic devices according to the variousembodiments of the invention. The active and passive functionalizationareas are shaded differently as denoted. In particular, first channels(vias) and second channels that connect to them forming a so calledrevolver pattern are used in these examples to illustrate differentialfunctionalization in three dimensions. The specific layout of thethree-dimensional features within these exemplary substrates is largelyunimportant for the functionalization process, with the exception of afew guidelines that help control the application of resist.

FIG. 37 parts A-F illustrates an exemplary workflow for the generationof differential functionalization patterns illustrated in FIG. 37 partB-D. Accordingly, the substrate may first be cleaned, for example usinga piranha solution, followed by O₂ plasma exposure (FIG. 37 part A).Photoresist may be applied to the device layer embedding the secondchannels (aka revolvers; FIG. 37 part B). A photolithography and/or aplasma descum step may be used to generate a desired pattern ofphotoresist on the substrate, using a suitable mask for the pattern(FIG. 37 part C). The mask pattern may be varied to control where thephotoresist stays and where it is cleared. A functionalization step, forexample with a fluorosilane, a hydrocarbon silane, or any group formingan organic layer that may passivate the surface, may be performed todefine the passively functionalized areas on the device (FIG. 37 partD). The resist may be stripped using a suitable method describedelsewhere herein or otherwise known in the art (FIG. 37 part E). Oncethe resist is removed, the exposed areas may be subject to activefunctionalization leaving the desired differential functionalizationpattern (FIG. 37 part F).

In various embodiments, the methods and compositions described hereinrelate to the application of photoresist for the generation of modifiedsurface properties in selective areas, wherein the application of thephotoresist relies on the fluidic properties of the substrates definingthe spatial distribution of the photoresist. Without being bound bytheory, surface tension effects related to the applied fluid may definethe flow of the photoresist. For example surface tension and/orcapillary action effects may facilitate drawing of the photoresist intosmall structures in a controlled fashion before the resist solventsevaporate (FIG. 38). In one embodiment, resist contact points get pinnedby sharp edges, thereby controlling the advance of the fluid. Theunderlying structures may be designed based on the desired flow patternsthat are used to apply photoresist during the manufacturing andfunctionalization processes. A solid organic layer left behind aftersolvents evaporate may be used to pursue the subsequent steps of themanufacturing process.

Substrates may be designed to control the flow of fluids by facilitatingor inhibiting wicking effects into neighboring fluidic paths. Forexample, FIG. 39 part A illustrates a design avoiding overlap betweentop and bottom edges, which facilitates the keeping of the fluid in topstructures allowing for a particular disposition of the resist. Incontrast, FIG. 39 part B illustrates an alternative design, wherein thetop and bottom edges do overlap, leading to the wicking of the appliedfluid into bottom structures. Appropriate designs may be selectedaccordingly, depending on the desired application of the resist.

FIG. 40 illustrates bright field (part A) and dark field (part B) imagesof a device that is subjected to resist according to the illustratedsmall disk photoresist pattern in FIG. 40 part C after photolithography.

FIG. 41 illustrates bright field (part A) and dark field (part B) imagesof a device that is subjected to resist according to the illustratedfull disk photoresist pattern in FIG. 41 part 41C afterphotolithography.

FIG. 42 illustrates bright field (part A) and dark field (part B) imagesof a device that is functionalized according to the pattern in FIG. 42part C after passive functionalization and stripping of the resist.

FIG. 43 illustrates the differing fluidic properties of thedifferentially functionalized surfaces in bright field (part A) and darkfield (part B) images according to the pattern in FIG. 43 part C usingdimethylsulfoxide (DMSO) as a fluid. Spontaneous wetting of therevolvers was achieved using the hydrophilic surfaces within therevolvers surrounded by the hydrophobic areas.

FIG. 44 illustrates another exemplary workflow for the generation ofdifferential functionalization patterns illustrated in FIG. 36 part F.Accordingly, the substrate may first be cleaned, for example using apiranha solution, followed by O₂ plasma exposure (FIG. 44 part 44A). Afunctionalization step, for example with a fluorosilane, a hydrocarbonsilane, or any group that can form an organic layer that may passivatethe surface, may be performed to define the passively functionalizedareas on the device (FIG. 44 part B). Photoresist may be applied to thedevice layer embedding the second channels (aka revolvers; FIG. 44 partC). A photolithography and/or an etch step may be used to generate adesired pattern of photoresist on the substrate, using a suitable maskfor the pattern (FIG. 44 part D). The mask pattern may be varied tocontrol where the photoresist stays and where it is cleared. The resistmay be stripped using a suitable method described elsewhere herein orotherwise known in the art (FIG. 44 part E). Once the resist is removed,the exposed areas may be subject to active functionalization leaving thedesired differential functionalization pattern (FIG. 44 part F).

In another embodiment, the functionalization workflow is designed suchthat the resist is applied from the via (bottom) side and flown into thevias and the revolvers. The exposed areas on the outer surfaces may besubjected to functionalization. The resist may be removed, for examplefrom the back (bottom) side of the device using lithography or etching,allowing active functionalization in the exposed areas leading to thepattern described in FIG. 36 part E.

In yet another embodiment, an overlap design may be chosen between thevias and the revolver channel edges as shown in FIG. 39 part B. Theresist may be applied from the front (top) side wicking the fluid intothe vias. Passive functionalization, stripping of the resist, followedby active functionalization would lead to the manufacturing of thepattern illustrated in FIG. 36 part E.

An exemplary microfluidic device comprising a substantially planarsubstrate portion is shown as a diagram in FIG. 25 part D. Across-section of the diagram is shown in FIG. 25 part E. The substratecomprises a plurality of clusters, wherein each cluster comprises aplurality of groupings of fluidic connections. Each grouping comprises aplurality of second channels extending from a first channel FIG. 25 partA is a device view of a cluster comprising a high density of groupings.FIG. 25 part C is a handle view of the cluster of FIG. 25A. FIG. 25 partB is a section view of FIG. 25 part A.

A cluster of groupings may be arranged in any number of conformations.In FIG. 25 part A, the groupings are arranged in offset rows to form acluster in a circle-like pattern. FIG. 25 part C depicts arrangement ofa plurality of such clusters on an exemplary microfluidic device. Insome embodiments, individual clusters are contained within individualcluster regions whose interior forms a convex set. In some embodiments,the individual cluster regions are non-overlapping with each other. Theindividual cluster regions may be a circle or any other suitablepolygon, e.g. a triangle, a square, a rectangle, a, a parallelogram, ahexagon etc. As represented by 2503, an exemplary distance between threerows of groupings may be from about 0.05 mm to about 1.25 mm, asmeasured from the center of each grouping. The distance between 2, 3, 4,5, or more rows of groupings may be about or at least about 0.05 mm, 0.1mm, 0.15 mm, 0.2 mm, 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm, 0.45 mm, 0.5 mm,0.55 mm, 0.6 mm, 0.65 mm, 0.7 mm, 0.75 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm,1.2 mm, 1.2 mm, or 1.3 mm. The distance between 2, 3, 4, 5, or more rowsof groupings may be about or at most about 1.3 mm, 1.2 mm, 1.1 mm, 1 mm,0.9 mm, 0.8 mm, 0.75 mm, 0.65 mm, 0.6 mm, 0.55 mm, 0.5 mm, 0.45 mm, 0.4mm, 0.35 mm, 0.3 mm, 0.25 mm, 0.2 mm, 0.15 mm, 0.1 mm, 0.05 mm or less.The distance between 2, 3, 4, 5, or more rows of groupings may rangebetween 0.05-1.3 mm, 0.1-1.2 mm, 0.15-1.1 mm, 0.2-1 mm, 0.25-0.9 mm,0.3-0.8 mm, 0.35-0.8 mm, 0.4-0.7 mm, 0.45-0.75 mm, 0.5-0.6 mm, 0.55-0.65mm, or 0.6-0.65 mm. Those of skill in the art appreciate that thedistance may fall within any range bound by any of these values, forexample 0.05 mm-0.8 mm. As shown by 2506, an exemplary distance betweentwo groupings in a row of groupings may be from about 0.02 mm to about0.5 mm, as measured from the center of each grouping. The distancebetween two groupings in a row of groupings may be about or at leastabout 0.02 mm, 0.04 mm, 0.06 mm, 0.08 mm, 0.1 mm, 0.12 mm, 0.14 mm, 0.16mm, 0.18 mm, 0.2 mm, 0.22 mm, 0.24 mm, 0.26 mm, 0.28 mm, 03 mm, 0.32 mm,0.34 mm, 0.36 mm, 0.38 mm, 04 mm, 0.42 mm, 0.44 mm, 0.46 mm, 0.48 mm or0.5 mm. The distance between two groupings in a row of groupings may beabout or at most about 0.5 mm, 0.48 mm, 0.46 mm, 0.44 mm, 0.42 mm, 0.4mm, 0.38 mm, 0.36 mm, 0.34 mm, 0.32 mm, 0.3 mm, 0.28 mm, 0.26 mm, 0.24mm, 0.22 mm, 0.2 mm, 0.18 mm, 0.16 mm, 0.14 mm, 0.12 mm, 0.1 mm, 0.08mm, 0.06 mm, 0.04 mm, or 0.2 mm or less. The distance between twogroupings may range between 0.02-0.5 mm, 0.04-0.4 mm, 0.06-0.3 mm, or0.08-0.2 mm. Those of skill in the art appreciate that the distance mayfall within any range bound by any of these values, for example 0.04mm-0.2 mm.

The length and width of the first and second channels of each groupingmay be optimized according to experimental conditions. In someembodiments, the cross-section of a first channel in a grouping,represented by 2504, is about or at least about 0.01 mm, 0.015 mm, 0.02mm, 0.025 mm, 0.03 mm, 0.035 mm, 0.04 mm, 0.045 mm, 0.05 mm, 0.055 mm,0.06 mm, 0.065 mm, 0.07 mm, 0.075 mm, 0.08 mm, 0.085 mm, 0.09 mm, 0.1mm, 0.15 mm, 0.2 mm, 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm, 0.45 mm, or 0.5mm. In some embodiments, the cross-section of a first channel in agrouping is about or at most about 0.5 mm, 0.45 mm, 0.4 mm, 0.35 mm, 0.3mm, 0.25 mm, 0.2 mm, 0.15 mm, 0.1 mm, 0.09 mm, 0.085 mm, 0.08 mm, 0.075mm, 0.07 mm, 0.065 mm, 0.06 mm, 0.055 mm, 0.05 mm, 0.045 mm, 0.04 mm,0.035 mm, 0.03 mm, 0.025 mm, 0.02 mm, 0.015 mm, or 0.01 mm or less. Thecross-section of a first channel in a grouping may range between0.01-0.5 mm, 0.02-0.45 mm, 0.03-0.4 mm, 0.04-0.35 mm, 0.05-0.3 mm,0.06-0.25, or 0.07-0.2 mm. Those of skill in the art appreciate that thedistance may fall within any range bound by any of these values, forexample 0.04 mm-0.2 mm. In some embodiments, the cross-section of asecond channel in a grouping, represented by 2505, is about or at leastabout 0.001 mm, 0.002 mm, 0.004 mm, 0.006 mm, 0.008 mm, 0.01 mm, 0.012mm, 0.014 mm, 0.016 mm, 0.018 mm, 0.02 mm, 0.025 mm, 0.03 mm, 0.035 mm,0.04 mm, 0.045 mm, 0.05 mm, 0.055 mm, 0.06 mm, 0.065 mm, 0.07 mm, 0.075mm, or 0.08 mm. In some embodiments, the cross-section of a secondchannel in a grouping, is about or at most about 0.08 mm, 0.075 mm, 0.07mm, 0.065 mm, 0.06 mm, 0.055 mm, 0.05 mm, 0.045 mm, 0.04 mm, 0.035 mm,0.03 mm, 0.025 mm, 0.02 mm, 0.018 mm, 0.016 mm, 0.014 mm, 0.012 mm, 0.01mm, 0.008 mm, 0.006 mm, 0.004 mm, 0.002 mm, 0.001 mm or less. Thecross-section of a second channel in a grouping may range between0.001-0.08 mm, 0.004-0.07 mm, 0.008-0.06 mm, 0.01-0.05 mm, 0.015-0.04mm, 0.018-0.03 mm, or 0.02-0.025 mm. Those of skill in the artappreciate that the distance may fall within any range bound by any ofthese values, for example 0.008 mm-0.04 mm. FIG. 25 part B depicts anexemplary cross-section of a cluster comprising a row of 11 groupings.In some embodiments, the height of the second channel in each groupingis about or at least about 0.005 mm, 0.008 mm, 0.01 mm, 0.015 mm, 0.02mm, 0.025 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.1mm, 0.12 mm, 0.14 mm, 0.16 mm, 0.18 mm, or 0.2 mm long. In someembodiments, the height of the second channel, shown as 2501, in eachgrouping is about or at most about 0.2 mm, 0.18 mm, 0.16 mm, 0.14 mm,0.12 mm, 0.1 mm, 0.08 mm, 0.07 mm, 0.06 mm, 0.05 mm, 0.04 mm, 0.03 mm,0.025 mm, 0.02 mm, 0.015 mm, 0.01 mm, 0.008 mm, or 0.005 mm long. Theheight of the second channel in each grouping may range between0.005-0.2 mm, 0.008-0.18 mm, 0.01-0.16 mm, 0.015-0.1 mm, 0.02-0.08 mm,or 0.025-0.04 mm. Those of skill in the art appreciate that the distancemay fall within any range bound by any of these values, for example 0.01mm-0.04 mm. In some embodiments, the height of the first channel withineach grouping, shown as 2502, is about or at most about 5 mm, 4.5 mm, 4mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1.0 mm, 0.8 mm, 0.5 mm, 0.4 mm,0.375 mm, 0.35 mm, 0.3 mm, 0.275 mm, 0.25 mm, 0.225 mm, 0.2 mm, 0.175mm, 0.15 mm, 0.125 mm, 0.1 mm, 0.075 mm, or 0.05 mm. In someembodiments, the height of the first channel within each grouping, shownas 2502, is about or at least about 0.05 mm, 0.075 mm, 0.1 mm, 0.125 mm,0.15 mm, 0.175 mm, 0.2 mm, 0.225 mm, 0.25 mm, 0.275 mm, 0.3 mm, 0.325mm, 0.35 mm, 0.375 mm, 0.4 mm, 0.5 mm, 0.8 mm, 10 mm, 1.5 mm, 2 mm, 2.5mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm. The height of the first channelwithin each grouping may range between 0.05-5 mm, 0.075-4 mm, 0.1-3 mm,0.15-2 mm, 0.2-1 mm, or 0.3-0.8 mm. Those of skill in the art appreciatethat the distance may fall within any range bound by any of thesevalues, for example 0.1 mm-1 mm.

The cluster of groupings may be arranged in a conformation suitable forplacement in a single reaction well of the substantially planarsubstrate portion of a microfluidic device, as shown in FIG. 25 part D.FIG. 25 part D is a diagram of a substantially planar substrate portionof a microfluidic device comprising 108 reaction wells, wherein eachreaction well comprises a plurality of groupings. A substrate maycomprise any number of wells, including but not limited to, any numberbetween about 2 and about 250. In some embodiments, the number of wellsincludes from about 2 to about 225 wells, from about 2 to about 200wells, from about 2 to about 175 wells, from about 2 to about 150 wells,from about 2 to about 125 wells, from about 2 to about 100 wells, fromabout 2 to about 75 wells, from about 2 to about 50 wells, from about 2to about 25 wells, from about 25 to about 250 wells, from about 50 toabout 250 wells, from about 75 to about 250 wells, from about 100 toabout 250 wells, from about 125 to about 250 wells, from about 150 toabout 250 wells, from about 175 to about 250 wells, from about 200 toabout 250 wells, or from about 225 to about 250 wells. Those of skill inthe art appreciate that the well number may fall within any range boundby any of these values, for example 25-125. In addition, each well cancomprise a cluster of any number groupings, including, but not limitedto, any number between about 2 and about 250 groupings. In someembodiments, a cluster comprises from about 2 to about 225 groupings,from about 2 to about 200 groupings, from about 2 to about 175groupings, from about 2 to about 150 groupings, from about 2 to about125 groupings, from about 2 to about 100 groupings, from about 2 toabout 75 groupings, from about 2 to about 50 groupings, from about 2 toabout 25 groupings, from about 25 to about 250 groupings, from about 50to about 250 groupings, from about 75 to about 250 groupings, from about100 to about 250 groupings, from about 125 to about 250 groupings, fromabout 150 to about 250 groupings, from about 175 to about 250 groupings,from about 200 to about 250 groupings, or from about 225 to about 250groupings. Those of skill in the art appreciate that the number ofgroupings may fall within any range bound by any of these values, forexample 25-125. As an example, each of the 108 wells of the substrateshown in FIG. 25 part D, can comprise a cluster of 109 groupings shownin FIG. 25 part A, resulting in 11,772 groupings present in thesubstantially planar substrate portion of the microfluidic device.

FIG. 25 part D includes an origin of reference indicated by a 0,0 (X,Y)axis, wherein the bottom left corner of an exemplary substantiallyplanar substrate portion of a microfluidic device is diagramed. In someembodiments, the width of the substantially planar substrate,represented as 2508, is from about 5 mm to about 150 mm along onedimension, as measured from the origin. In some embodiments, the widthof a substantially planar substrate, represented as 2519, is from about5 mm to about 150 mm along another dimension, as measured from theorigin. In some embodiments, the width of a substrate in any dimensionis from about 5 mm to about 125 mm, from about 5 mm to about 100 mm,from about 5 mm to about 75 mm, from about 5 mm to about 50 mm, fromabout 5 mm to about 25 mm, from about 25 mm to about 150 mm, from about50 mm to about 150 mm, from about 75 mm to about 150 mm, from about 100mm to about 150 mm, or from about 125 mm to about 150 mm. Those of skillin the art appreciate that the width may fall within any range bound byany of these values, for example 25-100 mm. The substantially planarsubstrate portion shown in FIG. 25 part D comprises 108 clusters ofgroupings. The clusters may be arranged in any configuration. In FIG. 25part D, the clusters are arranged in rows forming a square shape.Regardless of arrangement, the clusters may start at a distance of about0.1 mm to about 149 mm from the origin, as measured on the X- or Y-axis.Lengths 2518 and 2509 represent the furthest distances of the center ofa cluster on the X- and Y-axis, respectively. Lengths 2517 and 2512represent the closest distances of the center of a cluster on the X- andY-axis, respectively. In some embodiments, the clusters are arranged sothat there exists a repeated distance between two clusters. As shown by2507 and 2522, the distance between two clusters may be from about 0.3mm to about 9 mm apart. In some embodiments, the distance between twoclusters is about or at least about 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7mm, 0.8 mm 0.9 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm,2.4 mm, 2.6 mm, 2.8 mm, 3 mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2mm, 4.4 mm, 4.6 mm, 4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm,6.2 mm, 6.4 mm, 6.6 mm, 6.8 mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8mm, 8.2 mm, 8.4 mm, 8.6 mm, 8.8 mm, or 9 mm. In some embodiments, thedistance between two clusters is about or at most about 9 mm, 8.8 mm,8.6 mm, 8.4 mm, 8.2 mm, 8 mm, 7.8 mm, 7.6 mm, 7.4 mm, 7.2 mm, 7 mm, 6.8mm, 6.6 mm, 6.4 mm, 6.2 mm, 6 mm, 5.8 mm, 5.6 mm, 5.4 mm, 5.2 mm, 5 mm,4.8 mm, 4.6 mm, 4.4 mm, 4.2 mm, 4 mm, 3.8 mm, 3.6 mm, 3.4 mm, 3.2 mm, 3mm, 2.8 mm, 2.6 mm, 2.4 mm, 2.2 mm, 2 mm, 1.8 mm, 1.6 mm, 1.4 mm, 1.2mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, or 0.3 mm. Thedistance between two clusters may range between 0.3-9 mm, 0.4-8 mm,0.5-7 mm, 0.6-6 mm, 0.7-5 mm, 0.7-4 mm, 0.8-3 mm, or 0.9-2 mm. Those ofskill in the art appreciate that the distance may fall within any rangebound by any of these values, for example 0.8 mm-2 mm.

Fiducial marks may be placed on microfluidic devices described herein tofacilitate alignment of such devices with other components of a system.Microfluidic devices of the invention may have one or more fiducialmarks, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fiducial marks. Thesubstantially planar substrate portion of an exemplary microfluidicdevice shown in FIG. 25 part D comprises three fiducial marks useful foraligning the device with other components of a system. A fiducial markmay be located at any position within the substantially planar substrateportion of the microfluidic device. As shown by 2513 and 2516, afiducial mark may be located near the origin, where the fiducial mark iscloser to the origin than any one cluster. In some embodiments, afiducial mark is located near an edge of the substrate portion, as shownby 2511 and 2521, where the distance from the edge is indicated by 2510and 2520, respectively. The fiducial mark may be located from about 0.1mm to about 10 mm from the edge of the substrate portion. In someembodiments, the fiducial mark is located about or at least about 0.1mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm 0.9 mm, 1 mm,1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6 mm,6.8 mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm, 8.6mm, 8.8 mm, 9 mm, or 10 mm from the edge of the substrate portion. Insome embodiments, the fiducial mark is located about or at most about 10mm, 9 mm, 8.8 mm, 8.6 mm, 8.4 mm, 8.2 mm, 8 mm, 7.8 mm, 7.6 mm, 7.4 mm,7.2 mm, 7 mm, 6.8 mm, 6.6 mm, 6.4 mm, 6.2 mm, 6 mm, 5.8 mm, 5.6 mm, 5.4mm, 5.2 mm, 5 mm, 4.8 mm, 4.6 mm, 4.4 mm, 4.2 mm, 4 mm, 3.8 mm, 3.6 mm,3.4 mm, 3.2 mm, 3 mm, 2.8 mm, 2.6 mm, 2.4 mm, 2.2 mm, 2 mm, 1.8 mm, 1.6mm, 1.4 mm, 1.2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4mm, 0.3 mm, 0.2 mm, or 0.1 mm from the substrate portion. The fiducialmark may be located between 0.1-10 mm, 0.2-9 mm, 0.3-8 mm, 0.4-7 mm,0.5-6 mm, 0.1-6 mm, 0.2-5 mm, 0.3-4 mm, 0.4-3 mm, or 0.5-2 mm from theedge of the substrate. Those of skill in the art appreciate that thedistance may fall within any range bound by any of these values, forexample 0.1 mm-5 mm. The fiducial mark may be located close in distanceto a cluster, where exemplary X- and Y-axis distances are indicated by2515 and 2514, respectively. In some embodiments, a distance between acluster and a fiducial mark is about or at least about 0.001 mm, 0.005mm, 0.01 mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08mm, 0.09 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8mm, 0.9 mm, 1 mm, 1.2 mm, 1.5 mm, 1.7 mm, 2 mm, 2.2 mm, 2.5 mm, 2.7 mm,3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, or 8 mm. In someembodiments, a distance between a cluster and a fiducial mark is aboutor at most about 8 mm, 6.5 mm, 6 mm, 5.5 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm,3 mm, 2.7 mm, 2.5 mm, 2.2 mm, 2 mm, 1.7 mm, 1.5 mm, 1.2 mm, 1 mm, 0.9mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.09mm, 0.08 mm, 0.07 mm, 0.06 mm, 0.05 mm, 0.04 mm, 0.03 mm, 0.02 mm, 0.01mm, 0.005 mm, or 0.001 mm. The distance between a cluster and a fiducialmark may be in a range between 0.001-8 mm, 0.01-7 mm, 0.05-6 mm, 0.1-5mm, 0.5-4 mm, 0.6-3 mm, 0.7-2 mm, or 0.8-1.7 mm. Those of skill in theart appreciate that the distance may fall within any range bound by anyof these values, for example 0.5-2 mm.

FIG. 25 part E depicts a cross section of the substantially planarsubstrate portion of an exemplary microfluidic device shown in FIG. 25part D. The section shows a row of 11 groupings, each comprising acluster of groupings, wherein each grouping comprises a plurality ofsecond channels extending from a first channel. As exemplified by 2523,the total length of a grouping may be from about 0.05 mm to about 5 mmlong. In some embodiments, the total length of a grouping is about or atleast about 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.1 mm, 0.2 mm,0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.2 mm,1.5 mm, 1.7 mm, 2 mm, 2.2 mm, 2.5 mm, 2.7 mm, 3 mm, 3.2 mm, 3.5 mm, 3.7mm, 4 mm, 4.2 mm, 4.5 mm, 4.7 mm, or 5 mm. In some embodiments, thetotal length of a grouping is about or at most about 5 mm, 4.7 mm, 4.5mm, 4.2 mm, 4 mm, 3.7 mm, 3.5 mm, 3.2 mm, 3 mm, 2.7 mm, 2.5 mm, 2.2 mm,2 mm, 1.7 mm, 1.5 mm, 1.2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.09 mm, 0.08 mm, 0.07 mm, 0.06 mm,or 0.05 mm or less. The total length of a grouping may be in a rangebetween 0.05-5 mm, 0.06-4 mm, 0.07-3 mm, 0.08-2 mm, 0.09-1 mm, 0.1-0.9mm, 0.2-0.8 mm, or 0.3-0.7 mm. Those of skill in the art appreciate thatthe distance may fall within any range bound by any of these values, forexample 0.1-0.7 mm. In some embodiments, the microfluidic device mayhave a location for a label or a serial label, as exemplified in FIG. 25part F depicting an exemplary layout of clusters in a microfluidicdevice. The label may be located near an edge of the substrate, asexemplified by the distance 2603. In some embodiments, the label islocated from about 0.1 mm to about 10 mm from the edge of the substrate.In some embodiments, the label is located about or at least about 0.1mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm 0.9 mm, 1 mm,1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6 mm,6.8 mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm, 8.6mm, 8.8 mm, 9 mm, or 10 mm from the edge of a substrate. In someembodiments, the label is located about or at most about 10 mm, 9 mm,8.8 mm, 8.6 mm, 8.4 mm, 8.2 mm, 8 mm, 7.8 mm, 7.6 mm, 7.4 mm, 7.2 mm, 7mm, 6.8 mm, 6.6 mm, 6.4 mm, 6.2 mm, 6 mm, 5.8 mm, 5.6 mm, 5.4 mm, 5.2mm, 5 mm, 4.8 mm, 4.6 mm, 4.4 mm, 4.2 mm, 4 mm, 3.8 mm, 3.6 mm, 3.4 mm,3.2 mm, 3 mm, 2.8 mm, 2.6 mm, 2.4 mm, 2.2 mm, 2 mm, 1.8 mm, 1.6 mm, 1.4mm, 1.2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3mm, 0.2 mm, or 0.1 mm from the edge of a substrate. The distance may bein a range between 0.1-10 mm, 0.2-9 mm, 0.3-8 mm, 0.4-7 mm, 0.5-6 mm,0.6-5 mm, 0.7-4 mm, 0.8-3 mm, 0.9-2 mm or 1.5 mm. Those of skill in theart appreciate that the distance may fall within any range bound by anyof these values, for example 0.5-2 mm. The label may start at a positionfrom about 0.1 mm to about 20 mm from the origin as exemplified by 2602.The label may have a length from about 1 mm to about 32 mm asexemplified by 2601.

Wafers with Large Sized Vias for High Mass Oligonucleotide Synthesis

In some embodiments, the invention provides for methods and systems forcontrolled flow and mass transfer paths for oligonucleotide synthesis ona surface. The advantages of the systems and methods provided hereinallow for improved levels of structure for the controlled and evendistribution of mass transfer paths, chemical exposure times, and washefficacy during oligonucleotide synthesis. Further, the methods andsystems described herein allow for increased sweep efficiency, such asby providing sufficient volume for a growing oligonucleotide such thatthe excluded volume by the growing oligonucleotide does not take up morethan 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, 1%, or less of the initially available volume that is availableor suitable for growing oligonucleotides. In addition, the methods andsystems described herein allow for an sufficient structure for thegrowth of oligomers beyond 80 mer to 100, 120, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500-mer or longer.

Accordingly, the methods and systems described herein provide solutionsto achieve these advantages, such as collections of small parallelpassages. Structures, such as small vias may be used to feed smallerstructures, such as those found in the “revolver pattern” (FIG. 56B).Structures having a low surface energy surface on the inner surface maycause gas to hang up on the walls. Gas bubbles may impede the flow rateand flow uniformity during oligonucleotide synthesis cycles orsubsequent aqueous steps used for gene assembly. Accordingly, structuresthat are adapted for oligonucleotide synthesis may comprise a surfacewith increased surface energy as described elsewhere herein.

In some embodiments, the methods and systems of the invention exploitsilicon wafer processes for manufacturing substrates for oligonucleotidesynthesis. Such substrates may have a series of sites accessible tomaterial deposition via a deposition device such as an inkjet.Substrates manufactured according to the various embodiments of theinvention may support flood chemistry steps that are shared among aplurality of such sites through their plane. In various embodiments,devices allow aqueous reagents to be injected and pooled in a largerelief (FIG. 61 parts A-B).

In various embodiments, such oligonucleotide synthesis devices withlarge vias are created on a standard Silicon on Insulator (SOI) siliconwafer. The oligonucleotide synthesis device may have a total width of atleast or at least about 10 micrometer (μm), 11 μm, 12 μm, 13 μm, 14 μm,15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180μm, 190 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000μm, or more. The oligonucleotide synthesis device may have a total widthof at most or at most about 1000 μm, 900 μm, 850 μm, 750 μm, 700 μm, 650μm, 600 μm, 550 μm, 500 μm, 450 μm, 400 μm, 350 μm, 300 μm, 250 μm, 200μm, 190 μm, 180 μm, 170 μm, 160 μm, 150 μm, 140 μm, 130 μm, 120 μm, 110μm, 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 19 μm, 18 μm, 17μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, or less. Theoligonucleotide synthesis device may have a total width that is between10-1000 μm, 11-950 μm, 12-900 μm, 13-850 μm, 14-800 μm, 15-750 μm,16-700 μm, 17-650 μm, 18-600 μm, 19-550 μm, 20-500 μm, 25-450 μm, 30-400μm, 35-350 μm, 40-300 μm, 45-250 μm, 50-200 μm, 55-150 μm, 60-140 μm,65-130 μm, 70-120 μm, 75-110 μm, 70-100 μm, 75-80 μm, 85-90 μm or 90-95μm. Those of skill in the art appreciate that the total width of theoligonucleotide synthesis device may fall within any range bound by anyof these values, for example 20-80 μm. The total width of theoligonucleotide device may fall within any range defined by any of thevalues serving as endpoints of the range. It may be subdivided into ahandle layer and a device layer. All or portions of the device may becovered with a silicon dioxide layer. The silicon dioxide layer may havea thickness of at least or at least about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm,6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm,45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9μm, 2.0 μm, or more. The silicon dioxide layer may have a thickness ofat most or at most about 2.0 μm, 1.9 μm, 1.8 μm, 1.7 μm, 1.6 μm, 1.5 μm,1.4 μm, 1.3 μm, 1.2 μm, 1.1 μm, 1.0 μm, 500 nm, 400 nm, 300 nm, 200 nm,175 nm, 150 nm, 125 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20nm, 15 nm, 10 nm, 9 nm, 8, nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm,or less. The silicon diooxide layer may have a thickness that is between1.0 nm-2.0 μm, 1.1-1.9 μm, 1.2-1.8 nm, 1.3-1.7 μm, 1.4-1.6 μm. Those ofskills in the art will appreciate that the silicon diooxide layer mayhave a thickness that falls within any range bound by any of thesevalues, for example (1.5-1.9 μm). The silicon dioxide may have athickness that falls within any range defined by any of the valuesserving as endpoints of the range.

The device layer may comprise a plurality of structures suitable foroligonucleotide growth, as described elsewhere herein, such as aplurality of small holes (FIG. 61 parts A-B). The device layer may havea thickness of at least or at least about 1 micrometer (μm), 2 μm, 3 μm,4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm,15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, or more. The device layermay have a thickness of at most or at most about 500 μm, 400 μm, 300 μm,200 μm, 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm,55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 19 μm, 18 μm, 17μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, or less. The device layer may have athickness that is between 1-100 μm, 2-95 μm, 3-90 μm, 4-85 μm, 5-80 μm,6-75 μm, 7-70 μm, 8-65 μm, 9-60 μm, 10-55 μm, 11-50 μm, 12-45 μm, 13-40μm, 14-35 μm, 15-30 μm, 16-25 μm, 17-20 μm, 18-19 μm. Those of skill inthe art appreciate that the thickness of the device layer may fallwithin any range bound by any of these values, for example (20-60 μm).The thickness of the device layer may fall within any range defined byany of the values serving as endpoints of the range. The handle and/orthe device layer may comprise deep features. Such deep features may bemanufactured using a suitable MEMS technique, such as deep reactive ionetching. A series of etches may be used to construct the desired devicegeometry. One of the etches may be allowed to last longer and penetratethe insulator layer. Accordingly, passages that span the entire width ofthe device may be constructed. Such passages may be used to pass fluidfrom one surface of a substrate, such as a substantially planarsubstrate, to another.

In some embodiments, the device layer has at least two and up to 500sites, from at least 2 to about 250 sites, from at least 2 to about 200sites, from at least 2 to about 175 sites, from at least 2 to about 150sites, from at least 2 to about 125 sites, from at least 2 to about 100sites, from at least 2 to about 75 sites, from at least 2 to about 50sites, from at least 2 to about 25 sites, or from at least 2 to about250 sites that penetrate through the device layer. In some embodiments,the device layer has at least or at least about 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,350, 400, 450, 500, or more sites. Those of skill in the art appreciatethat the number of sites that penetrate through the device layer mayfall within any range bound by any of these values, for example 75-150sites. The device layer may be at least or at least about 2 μm, 3 μm, 4μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95μm, 100 μm thick, or more. The device layer may be at most or at mostabout 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 19 μm, 18 μm, 17μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, thick, or less. The device layer canhave any thickness that fall between 1-100 μm, 2-95 μm, 3-90 μm, 4-85μm, 5-80 μm, 6-75 μm, 7-70 μm, 8-65 μm, 9-60 μm, 10-55 μm, 11-50 μm,12-45 μm, 13-40 μm, 14-35 μm, 15-30 μm, 16-25 μm, 17-20 μm, 18-19 μm.Those skilled in the art appreciate that the device layer can have anythickness that may fall within any range bound by any of these valuesbound by any of these values, for example, 4-100 μm.

The thickness of the device layer may fall within any range defined byany of the values serving as endpoints of the range. The handle layermay have a larger area etched into the wafer that neighbors the featuresin the device layer. The handle layer may have a thickness of at leastor at least about 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 110μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm, or more.The handle layer may have a thickness of at most or at most about 1000μm, 950 μm, 900 μm, 850 μm, 800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550μm, 500 μm, 450 μm, 400 μm, 350 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50μm, 45 μm, 40 μm, 30 μm, 25 μm, 20 μm, 19 μm, 18 μm, 17 μm, 16 μm, 15μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4μm, 3 μm, 2 μm, 1 μm, or less. The handle layer can have any thicknessthat is between 10-1000 μm, 11-950 μm, 12-900 μm, 13-850 μm, 14-800 μm,15-750 μm, 16-700 μm, 17-650 μm, 18-600 μm, 19-550 μm, 20-500 μm, 25-450μm, 30-400 μm, 35-350 μm, 40-300 μm, 45-250 μm, 50-200 μm, 55-150 μm,60-140 μm, 65-130 μm, 70-120 μm, 75-110 μm, 70-100 μm, 75-80 μm, 85-90μm or 90-95 μm. Those of skill in the art appreciate that handle layermay have a thickness that falls within any range bound by any of thesevalues, for example 20-350 μm. The thickness of the handle layer fallwithin any range defined by any of the values serving as endpoints ofthe range

Etched regions in the handle layer may form well-like structuresembedded in the substrate. In some embodiments, etched regions withinthe handle layer may have a thickness of at least or about at least 100μm, 101 μm, 102 μm, 103 μm, 104 μm, 105 μm, 106 μm, 107 μm, 108 μm, 109μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, or 1000 μm,or more. The etched region within the handle layer may have anythickness of at most or about at most 1000 μm, 950 μm, 900 μm, 850 μm,800 μm, 750 μm, 700 μm, 650 μm, 600 μm, 550 μm, 500 μm, 450 μm, 400 μm,350 μm, 300 μm, 250 μm, 200 μm, 190 μm, 180 μm, 170 μm, 160 μm, 150 μm,140 μm, 130 μm, 120 μm, 110 μm, 109 μm, 108 μm, 107 μm, 106 μm, 105 μm,104 μm, 103 μm, 102 μm, 101 μm, 100 μm, or less. The etched regionwithin the handle layer may have any thickness that is between 100-1000μm, 101-950 μm, 102-900 μm, 103-850 μm, 104-800 μm, 105-750 μm, 106-700μm, 105-650 μm, 106-600 μm, 107-550 μm, 108-500 μm, 109-450 μm, 110-400μm, 120-350 μm, 130-300 μm, 140-250 μm, 150-200 μm, 160-190 μm, 170-180μm. Those of skill in the art appreciate that handle layer may have athickness that falls within any range bound by any of these values, forexample 200-300 μm.

The shape of the etched regions within the handle layer may berectangular or curvilinear.

In some embodiments, large etched regions within the handle layer allowfor easy transition from a gas phase to a liquid phase during theoligonucleotide synthesis cycle, and/or during oligonucleotide release,such as oligonucleotide release into gas phase.

Substrates with High Surface Area Synthesis Sites

In various embodiments, the methods and systems described herein relateto oligonucleotide synthesis devices for the synthesis of high masses ofoligonucleotides. The synthesis may be in parallel. For example at leastor about at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,1000, 10000, 50000, 100000 or more oligonucleotides can be synthesizedin parallel. The total number oligonucleotides that may be synthesizedin parallel may be between 2-100000, 3-50000, 4-10000, 5-1000, 6-900,7-850, 8-800, 9-750, 10-700, 11-650, 12-600, 13-550, 14-500, 15-450,16-400, 17-350, 18-300, 19-250, 20-200, 21-150, 22-100, 23-50, 24-45,25-40, 30-35. Those of skill in the art appreciate that the total numberof oligonucleotides synthesized in parallel may fall within any rangebound by any of these values, for example 25-100. The total number ofoligonucleotides synthesized in parallel may fall within any rangedefined by any of the values serving as endpoints of the range. Totalmolar mass of oligonucleotides synthesized within the device or themolar mass of each of the oligonucleotides may be at least or at leastabout 10, 20, 30, 40, 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000picomoles, or more. The length of each of the oligonucleotides oraverage length of the oligonucleotides within the device may be at leastor about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200,300, 400, 500 nucleotides, or more. The length of each of theoligonucleotides or average length of the oligonucleotides within thedevice may be at most or about at most 500, 400, 300, 200, 150, 100, 50,45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 nucleotides,or less. The length of each of the oligonucleotides or average length ofthe oligonucleotides within the device may fall between 10-500, 9-400,11-300, 12-200, 13-150, 14-100, 15-50, 16-45, 17-40, 18-35, 19-25. Thoseof skill in the art appreciate that the length of each of theoligonucleotides or average length of the oligonucleotides within thedevice may fall within any range bound by any of these values, forexample 100-300. The length of each of the oligonucleotides or averagelength of the oligonucleotides within the device may fall within anyrange defined by any of the values serving as endpoints of the range.

In various embodiments, high surface areas are achieved by structuringthe surface of a substrate with raised and/or lower features asexemplified in FIG. 62. The raised or lowered features may have sharp orrounded edges and may have cross-sections (widths) of any desiredgeometric shape, such as rectangular, circular, etc. They may formchannels along the entire substrate surface or a portion of it. Theraised or lowered features may have an aspect ratio of at least or aboutat least 1:20, 2:20, 3:20, 4:20, 5:20, 6:20, 10:20, 15:20, 20:20, 20:10,20:5, 20:1, or more. The raised or lowered features may have an aspectratio of at most or about at most 20:1, 20:5, 20:10, 20:20, 20:15,20:10, 20:10, 6:20, 5:20, 4:20, 3:20, 2:20, 1:20, or less. The raised orlowered features may have an aspect ratio that falls between 1:20-20:1,2:20-20:5, 3:20-20:10, 4-20:20:15, 5:20-20:20, 6:20-20:20. Those ofskill in the art appreciate that the raised or lowered features may havean aspect ratio that may fall within any range bound by any of thesevalues, for example 3:20-4:20. The raised or lowered features may havean aspect ratio that falls within any range defined by any of the valuesserving as endpoints of the range.

The raised or lowered features may have cross-sections of at least orabout at least 10 nanometers (nm), 11 nm, 12 nm, 20 nm, 30 nm, 100 nm,500 nm, 1000 nm, 10000 nm, 100000 nm, 1000000 nm, or more. The raised orlowered features may have cross-sections of at least or most or about atmost 1000000 nm, 100000 nm, 10000 nm, 1000 nm, 500 nm, 100 nm, 30 nm, 20nm, 12 nm, 11 nm, 10 nm, or less. The raised or lowered features mayhave cross-sections that fall between 10 nm-1000000 nm, 11 nm-100000 nm,12 nm-10000 nm, 20 nm-1000 nm, 30 nm-500 nm. Those of skill in the artappreciate that the raised or lowered features may have cross-sectionsthat may fall within any range bound by any of these values, for example10 nm-100 nm. The raised or lowered features may have cross-sectionsthat fall within any range defined by any of the values serving asendpoints of the range.

The raised or lowered features may have heights of at least or about atleast 10 nanometers (nm), 11 nm, 12 nm, 20 nm, 30 nm, 100 nm, 500 nm,1000 nm, 10000 nm, 100000 nm, 1000000 nm, or more. The raised or loweredfeatures may have heights of at most or about at most 1000000 nanometers(nm), 100000 nm, 10000 nm, 1000 nm, 500 nm, 100 nm, 30 nm, 20 nm, 12 nm,11 nm, 10 nm, or less. The raised or lowered features may have heightsthat fall between 10 nm-1000000 nm, 11 nm-100000 nm, 12 nm-10000 nm, 20nm-1000 nm, 30 nm-500 nm. Those of skill in the art appreciate that theraised or lowered features may have heights that may fall within anyrange bound by any of these values, for example 100 nm-1000 nm. Theraised or lowered features may have heights that fall within any rangedefined by any of the values serving as endpoints of the range. Theindividual raised or lowered features may be separated from aneighboring raised or lowered feature by a distance of at least or atleast about 5 nanometers (nm), 10 nm, 11 nm, 12 nm, 20 nm, 30 nm, 100nm, 500 nm, 1000 nm, 10000 nm, 100000 nm, 1000000 nm, or more. Theindividual raised or lowered features may be separated from aneighboring raised or lowered feature by a distance of at most or aboutat most 1000000 nanometers (nm), 100000 nm, 10000 nm, 1000 nm, 500 nm,100 nm, 30 nm, 20 nm, 12 nm, 11 nm, 10 nm, 5 nm, or less. The raised orlowered features may have heights that fall between 5-1000000 nm,10-100000 nm, 11-10000 nm, 12-1000 nm, 20-500 nm, 30-100 nm. Those ofskill in the art appreciate that the individual raised or loweredfeatures may be separated from a neighboring raised or lowered featureby a distance that may fall within any range bound by any of thesevalues, for example 100-1000 nm. The individual raised or loweredfeatures may be separated from a neighboring raised or lowered featureby a distance that falls within any range defined by any of the valuesserving as endpoints of the range. In some embodiments, the distancebetween two raised or lowered features is at least or about at least0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0 times, or more, thecross-section (width) or average cross-section of the raised or loweredfeatures. The distance between the two raised or lowered features is atmost or about at most 10.0, 5.0, 3.0, 2.0, 1.0, 0.5, 0.2, 0.1 times, orless, the cross-section (width) or average cross-section of the raisedor lowered features. The distance between the two raised or loweredfeatures may be between 0.1-10, 0.2-5.0, 1.0-3.0 times, thecross-section (width) or average cross-section of the raised or loweredfeatures. Those of skill in the art appreciate that the distance betweenthe two raised or lowered features may be between any times thecross-section (width) or average cross-section of the raised or lowerfeatures within any range bound by any of these values, for example 5-10times. The distance between the two raised or lowered features may bewithin any range defined by any of the values serving as endpoints ofthe range.

In some embodiments, groups of raised or lowered features are separatedfrom each other. Perimeters of groups of raised or lowered features maybe marked by a different type of structural feature or by differentialfunctionalization. A group of raised or lowered features may bededicated to the synthesis of a single oligonucleotide. A group ofraised of lowered features may span an area that is at least or about atleast 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 20 μm, 50 μm, 70 μm, 90μm, 100 μm, 150 μm, 200 μm, or wider in cross section. A group of raisedof lowered features may span an area that is at most or about at most200 μm, 150 μm, 100 μm, 90 μm, 70 μm, 50 μm, 20 μm, 15 μm, 14 μm, 13 μm,12 μm, 11 μm, 10 μm, or narrower in cross section. A group of raised oflowered features may span an area that is between 10-200 μm, 11-150 μm,12-100 μm, 13-90 μm, 14-70 μm, 15-50 μm, 13-20 μm, wide incross-section. Those of skill in art appreciate that a group of raisedof lowered features may span an area that falls within any range boundby any of these values, for example 12-200 μm. A group of raised oflowered features may span an area that fall within any range defined byany of the values serving as endpoints of the range.

In various embodiments, the raised or lowered features on a substrateincrease the total available area for oligonucleotide synthesis by atleast or at least about 1.1, 1.2, 1.3, 1.4, 2, 5, 10, 50, 100, 200, 500,1000 fold, or more. The raised or lowered features on a substrateincrease the total available area for oligonucleotide synthesis between1.1-1000, 1.2-500, 1.3-200, 1.4-100, 2-50, 5-10, fold. Those of skill inart appreciate that the raised or lowered features on a substrate mayincrease the total available area for oligonucleotide synthesis betweenany fold bound by any of these values, for example 20-80 fold. Theraised or lowered features on a substrate increase the total availablearea for oligonucleotide synthesis by a factor that may fall within anyrange defined by any of the values serving as endpoints of the range.

The methods and systems of the invention using large oligonucleotidesynthesis surfaces allow for the parallel synthesis of a number ofoligonucleotides with nucleotide addition cycles times of at most orabout at most 20 min, 15 min, 14 min, 13 min, 12 min, 11 min, 10 min, 1min, 40 sec, 30 sec, or less. The methods and systems of the inventionusing large oligonucleotide synthesis surfaces allow for the parallelsynthesis of a number of oligonucleotides with nucleotide additioncycles times between 30 sec-20 min, 40 sec-10 min, 1 min-10 min Those ofskill in art appreciate that the methods and systems of the inventionusing large oligonucleotide synthesis surfaces allow for the parallelsynthesis of a number of oligonucleotides with nucleotide additioncycles times between any of these values, for example 30 sec-10 min. Themethods and systems of the invention using large oligonucleotidesynthesis surfaces allow for the parallel synthesis of a number ofoligonucleotides with nucleotide addition cycles times that may be fallbetween any range defined by any of the values serving as endpoints ofthe range.

The overall error rate or error rates for individual types of errorssuch as deletions, insertions, or substitutions for each oligonucleotidesynthesized on the substrate, for at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the oligonucleotidessynthesized on the substrate, or for the substrate average may be atmost or at most about 1:100, 1:500, 1:1000, 1:10000, 1:20000, 1:30000,1:40000, 1:50000, 1:60000, 1:70000, 1:80000, 1:90000, 1:1000000, orless. The overall error rate or error rates for individual types oferrors such as deletions, insertions, or substitutions for eacholigonucleotide synthesized on the substrate, for at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of theoligonucleotides synthesized on the substrate, or the substrate averagemay fall between 1:100 and 1:10000, 1:500 and 1:30000. Those of skill inart, appreciate that the overall error rate or error rates forindividual types of errors such as deletions, insertions, orsubstitutions for each oligonucleotide synthesized on the substrate, forat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,99.5%, or more of the oligonucleotides synthesized on the substrate, orthe substrate average may fall between any of these values, for example1:500 and 1:10000. The overall error rate or error rates for individualtypes of errors such as deletions, insertions, or substitutions for eacholigonucleotide synthesized on the substrate, for at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of theoligonucleotides synthesized on the substrate, or the substrate averagemay fall between any range defined by any of the values serving asendpoints of the range.

Standard silicon wafer processes can be employed to create a substratethat will have a high surface area as described above and a managedflow, allowing rapid exchange of chemical exposure. The oligonucleotidesynthesis substrate can be created with a series of structures withsufficient separation to allow oligomer chains greater than at least orabout at least 20 mer, 25 mer, 30 mer, 50 mer, 100 mer, 200 mer, 250mer, 300 mer, 400 mer, 500 mer, or more to be synthesized withoutsubstantial influence on the overall channel or pore dimension, forexample due to excluded volume effects, as the oligonucleotide grows.The oligonucleotide synthesis substrate can be created with a series ofstructures with sufficient separation to allow oligomer chains greaterthan at most or about at most 500 mer, 200 mer, 100 mer, 50 mer, 30 mer,25 mer, 20 mer, or less to be synthesized without substantial influenceon the overall channel or pore dimension, for example due to excludedvolume effects, as the oligonucleotide grows. The oligonucleotidesynthesis substrate can be created with a series of structures withsufficient separation to allow oligomer chains that are at least or atleast about 20 mer, 50 mer, 75 mer, 100 mer, 125 mer, 150 mer, 175 mer,200 mer, 250 mer, 300 mer, 350 mer, 400 mer, 500 mer, or more to besynthesized without substantial influence on the overall channel or poredimension, for example due to excluded volume effects, as theoligonucleotide grows. Those of skill in the art appreciate that theoligonucleotide synthesis substrate can be created with a series ofstructures with sufficient separation to allow oligomer chains greaterthan between any of these values, for example, 20-300 mer200 mer to besynthesized without substantial influence on the overall channel or poredimension, for example due to excluded volume effects, as theoligonucleotide grows.

FIG. 62 shows an exemplary substrate according to the embodiments of theinvention with an array of structures. The distance between the featuresmay be greater than at least or about at least 5 nm, 10 nm, 20 nm, 100nm, 1000 nm, 10000 nm, 100000 nm, 1000000 nm, or more. The distancebetween the features may be greater than at most or about at most1000000 nm, 100000 nm, 10000 nm, 1000 nm, 100 nm, 20 nm, 10 nm, 5 nm, orless. The distance between the features may fall between 5-1000000 nm,10-100000 nm, 20-10000 nm, 100-1000 nm. Those of skill in the artappreciate that the distance between the features may fall between anyof these values, for example, 20-1000 nm. The distance between thefeatures may fall between any range defined by any of the values servingas endpoints of the range. In one embodiment, the distance between thefeatures is greater than 200 nm. The features may be created by anysuitable MEMS processes described elsewhere herein or otherwise known inthe art, such as a process employing a timed reactive ion etch process.Such semiconductor manufacturing processes can typically create featuresizes smaller than 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 25 nm, 20 nm, 10nm, 5 nm, or less. Those of skill in the art appreciate that the featuresize smaller than 200 nm can be between any of these values, forexample, 20-100 nm. The feature size can fall within any range definedby any of these values serving as endpoints of the range. In oneembodiment, an array of 40 um wide posts are etched with 30 um depth,which about doubles the surface area available for synthesis.

The arrays of raised or lowered features may be segregated allowingmaterial deposition of a phosphoramidite chemistry for highly complexand dense library generation. The segration may be achieved by largerstructures or by differential functionalization of the surfacegenerating active and passive regions for oligonucleotide synthesis.Alternatively, the locations for the synthesis of individualoligonucleotides may be separated from each other by creating regions ofcleavable and non-cleavable oligonucleotide attachments to the surfaceunder a certain condition. A device, such as an inkjet printer, may beused to deposit reagents to the individual oligonucleotide synthesislocations. Differential functionalization can also achieve alternatingthe hydrophobicity across the substrate surface, thereby creating watercontact angle effects that may cause beading or wetting of the depositedreagents. Employing larger structures can decrease splashing andcross-contamination of individual oligonucleotide synthesis locationswith reagents of the neighboring spots.

Reactors

In another aspect, an array of enclosures is described herein. The arrayof enclosures can comprise a plurality of resolved reactors comprising afirst substrate and a second substrate comprising reactor caps. In somecases, at least two resolved loci are contained in each reactor. Theresolved reactors may be separated with a releasable seal. The reactorscaps may retain the contents of the reactors upon release of the secondsubstrate from the first substrate. The plurality of resolved reactorscan be any suitable density at a density of at least 1 per mm². Theplurality of reactor caps can be coated with a moiety. The moiety can bea chemically inert or chemically active moiety. The moiety that iscoated onto the reactor caps can be a moiety that can minimize theattachment of the oligonucleotides. The types of chemical moieties aredescribed in further detail elsewhere herein.

In some embodiments, the reactor caps described herein may relate toenclosures with an open top on the surface of a capping elementsubstrate. For example, the reactor caps may resemble cylinders stickingout on top of the substrate surface. The inner diameter of the reactorcaps can be about, at least about, or less than about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 115, 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475 or 500 μm. The outer diameter ofthe reactor caps can be about, at least about, or less than about 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 115, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or 600 μm. Therim of the cylinder can have a width of about, at least about, or lessthan about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, or 400 μm. The height of the reactor capmeasured inside can be about, at least about, or less than about 0.1,0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90or 100 μm. FIG. 7 illustrates an exemplary embodiment of reactor caps ona capping element.

All or part of the reactor cap surfaces, such as the rim surface, may bemodified using suitable surface modification methods described infurther detail elsewhere herein and otherwise known in the art. In somecases, surface irregularities are engineered. Chemical surfacemodifications and irregularities may serve to adjust the water contactangle of the rim. Similar surface treatments may also be applied on thesurface of a substrate that is brought in close proximity to the reactorcaps forming a seal, e.g. a reversible seal. A capillary burst valve maybe utilized between the two surfaces as described in further detailelsewhere herein. The surface treatments can be useful in precisecontrol of such seals comprising capillary burst valves.

The reactor caps comprised in a substrate may be in any shape or designthat is known in the art. The reactor cap may contain a volume of cavitythat is capable of enclosing the contents of the reactors. The contentsof the reactors may stem from a plurality of resolved loci on anadjacent substrate. The reactor cap can be in circular, elliptical,rectangular or irregular shapes. The reactor cap may have sharp corners.In some cases, the reactor cap may have round corners to minimizeretaining any air bubble and to facilitate better mixing of the contentsof the reactors. The reactor cap can be fabricated in any shape,organization or design that allows controlled transfer or mixing of thecontents of the reactors. The reactor cap can be in similar design asthe resolved loci on the substrate as described in the instantapplication. In some embodiments, the reactor caps can be in a shapethat allows liquid to easily flow in without creating air bubbles. Insome embodiments, the reactor caps can have a circular shape, with adiameter that can be about, at least about, or less than about 1micrometers (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm,11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm,150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 250 μm, 300 μm, 350 μm,400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm or 750 μm. Thereactor caps may have a monodisperse size distribution, i.e. all of themicrostructures may have approximately the same width, height, and/orlength. Alternatively, the reactor caps of may have a limited number ofshapes and/or sizes, for example the reactor caps may be represented in2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or more distinct shapes, eachhaving a monodisperse size. In some embodiments, the same shape can berepeated in multiple monodisperse size distributions, for example, 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or more monodisperse sizedistributions. A monodisperse distribution may be reflected in aunimodular distribution with a standard deviation of less than 25%, 20%,15%, 10%, 5%, 3%, 2%, 1%, 0.1%, 0.05%, 0.01%, 0.001% of the mode orsmaller.

Each of the reactor caps can have any suitable area for carrying out thereactions according to various embodiments of the invention describedherein. In some cases, the plurality of reactor caps can occupy anysuitable percentage of the total surface area of the substrate. In someembodiments, the plurality of the reactor caps can occupy about, atleast about, or less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the surfaceof the substrate. In some embodiments, the reactor caps can occupyabout, at least about, or less than about 0.1 mm², 0.15 mm², 0.2 mm²,0.25 mm², 0.3 mm², 0.35 mm², 0.4 mm², 0.45 mm², 0.5 mm², 0.55 mm², 0.6mm², 0.65 mm², 0.7 mm², 0.75 mm², 0.8 mm², 0.85 mm², 0.9 mm², 0.95 mm²,1 mm², 2 mm², 3 mm², 4 mm², 5 mm², 6 mm², 7 mm², 8 mm², 9 mm², 10 mm²,11 mm², 12 mm², 13 mm², 14 mm², 15 mm², 16 mm², 17 mm², 18 mm², 19 mm²,20 mm², 25 mm², 30 mm², 35 mm², 40 mm², 50 mm², 75 mm², 100 mm², 200mm², 300 mm², 400 mm², 500 mm², 600 mm², 700 mm², 800 mm², 900 mm², 1000mm², 1500 mm², 2000 mm², 3000 mm², 4000 mm², 5000 mm², 7500 mm², 10000mm², 15000 mm², 20000 mm², 25000 mm², 30000 mm², 35000 mm², 40000 mm²,50000 mm², 60000 mm², 70000 mm², 80000 mm², 90000 mm², 100000 mm²,200000 mm², 300000 mm² of total area, or more. The resolved reactors,the resolved loci and the reactor caps can be in any density. In someembodiments, the surface can have a density of resolved reactors,resolved loci or reactor caps of about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 15, about20, about 25, about 30, about 35, about 40, about 50, about 75, about100, about 200, about 300, about 400, about 500, about 600, about 700,about 800, about 900, about 1000, about 1500, about 2000, about 3000,about 4000, about 5000, about 6000, about 7000, about 8000, about 9000,about 10000, about 20000, about 40000, about 60000, about 80000, about100000, or about 500000 sites per 1 mm². In some embodiments, thesurface has a density of resolved reactors, resolved loci or reactorcaps of at least about 50, at least 75, at least about 100, at leastabout 200, at least about 300, at least about 400, at least about 500,at least about 600, at least about 700, at least about 800, at leastabout 900, at least about 1000, at least about 1500, at least about2000, at least about 3000, at least about 4000, at least about 5000, atleast about 6000, at least about 7000, at least about 8000, at leastabout 9000, at least about 10000, at least about 20000, at least about40000, at least about 60000, at least about 80000, at least about100000, or at least about 500000 sites per 1 mm².

Taken in account the density of the resolved loci on an adjacentsubstrate surface, the density, distribution, and shape of the reactorcaps can be designed accordingly to be configured to align with apreferred number of resolved loci in each reactor. Each of the pluralityof resolved reactors can comprise a number of resolved loci. Forexample, without limitation, each reactor can comprise about, at leastabout, less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225,250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, or 1000 resolved loci. In some cases, each reactor cancomprise at least 100 resolved loci.

Comprised within the array of the plurality of enclosures, the resolvedloci or reactor caps can reside on microstructures that are fabricatedinto a support surface. The microstructures can be fabricated by anyknown methods in the art, as described in other paragraphs herein. Themicrostructures can be microchannels or microwells that have any shapeand design in 2D or 3D. The microstructures (e.g., microchannels ormicrowells) may comprise at least two channels in fluidic communicationwith each other. For example, the microchannels can be interconnected,allowing fluid to perfuse through with given condition, such as vacuumsuction. Individual microstructures may be individually addressable andresolved, such that the contents of two resolved loci are kept unmixed.The microchannels can comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10channels in fluidic communications in any combinations, allowingcontrolled mixing, communicating or distributing of the fluid. Theconnectivity of microchannels can be controlled by valve systems thatare known in the art of microfluidic design. For example, a fluidcontrol layer of substrate can be fabricated directly on top of thefluidic communicating layer of the substrate. Different microfluidicvalves systems are described in Marc A. Unger et al, “MonolithicMicrofabricated Valves and Pumps by Multilayer Soft Lithography,”Science, vol. 288, no. 7, pp. 113-116, April 2000, and David C. Duffy etal., “Rapid Prototyping of Microfluidic Systems inPoly(dimethylsiloxane),” Analytical Chemistry, vol. 70, no. 23, pp.4974-4984, December 1998.

Comprised within the array of the plurality of enclosures, the resolvedloci or reactor caps can reside on microstructures such as microchannelsor channels. The dimensions and designs of the microchannels of theresolved loci on the adjacent substrate surface are described elsewhereherein. The microstructures may comprise at least two channels that arein fluidic communications, wherein the at least two channels cancomprise at least two channels with different width. In some cases, theat least two channels can have the same width, or a combination of thesame or different width. For example, without limitation, the width ofthe channels or microchannels can be about, at least about, or less thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 100 μm. The channels or microchannels can haveany length that allows fluidic communications of the resolved loci. Atleast one channel can comprise a ratio of surface area to length, or aperimeter, of about, at least about, less than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 100 μm. At least one channel can have a cross-sectional area that isin a circular shape and can comprise a radius of the cross-sectionalarea of about, at least about, less than about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or100 μm.

As described herein, an array of enclosures can comprise a plurality ofresolved reactors comprising a first substrate and a second substratecomprising reactor caps. The resolved reactors can be formed bycombining or capping the second substrate onto the first substrate, andsealed together. The seal can be reversible or irreversible. Inpreferred embodiments, the seal is reversible or releasable. Uponsealing the resolved reactors, the content of reactors such asoligonucleotides or reagents needed for amplification or otherdownstream reactions can be released and mixed within the resolvedreactors. The resolved reactors can be separated with a releasable sealand wherein the reactors caps can retain all or a portion of thecontents of the reactors upon release of the second substrate from thefirst substrate. Depending on the materials of the first substrate andthe second substrate, the seal can be designed differently to allowreversible seal in between the first substrate and the second substrate,and forming the resolved reactors. The first substrate and the secondsubstrate can come in direct physical contact when forming the seal. Insome cases, the first substrate and the second substrate can come inclose proximity without their respective surfaces immediately around ananoreactor or between two nanoreactors making a direct physicalcontact. The seal can comprise a capillary burst valve. The distance inbetween the first substrate and the second substrate when forming theseal can be about, at least about, less than about 0.1 μm, 0.2 μm, 0.3μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2μm, 1.3 μm, 1.4 μm, 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2 μm, 2.5μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm or 10 μm. The seal can comprise acapillary burst valve.

In some cases, the resolved enclosures may comprise pressure releaseholes. The pressure release holes may allow separation of the firstsubstrate and the second substrate. Design of microfluidic systems withpressure release system are described in European Patent No. EP 1987275A1, which is herein incorporated by reference in its entirety.

The plurality of resolved reactor caps on a substrate can bemanufactured by any method that is described herein or otherwise knownin the art (e.g., microfabrication processes). Microfabricationprocesses that may be used in making the substrate with the plurality ofreactor caps or reactors disclosed herein include without limitationlithography; etching techniques such as wet chemical, dry, andphotoresist removal; microelectromechanical (MEMS) techniques includingmicrofluidics/lab-on-a-chip, optical MEMS (also called MOEMS), RF MEMS,PowerMEMS, and BioMEMS techniques and deep reactive ion etching (DRIE);nanoelectromechanical (NEMS) techniques; thermal oxidation of silicon;electroplating and electroless plating; diffusion processes such asboron, phosphorus, arsenic, and antimony diffusion; ion implantation;film deposition such as evaporation (filament, electron beam, flash, andshadowing and step coverage), sputtering, chemical vapor deposition(CVD), epitaxy (vapor phase, liquid phase, and molecular beam),electroplating, screen printing, and lamination. See generally Jaeger,Introduction to Microelectronic Fabrication (Addison-Wesley PublishingCo., Reading Mass. 1988); Runyan, et al., Semiconductor IntegratedCircuit Processing Technology (Addison-Wesley Publishing Co., ReadingMass. 1990); Proceedings of the IEEE Micro Electro Mechanical SystemsConference 1987-1998; Rai-Choudhury, ed., Handbook of Microlithography,Micromachining & Microfabrication (SPIE Optical Engineering Press,Bellingham, Wash. 1997).

In an aspect, a substrate having a plurality of resolved reactor capscan be manufactured using any method known in the art. In someembodiments, the material of the substrate having a plurality of reactorcaps can be a semiconductor substrate such as silicon dioxide. Thematerials of the substrate can also be other compound III-V or II-VImaterials, such as (GaAs), a semiconductor produced via the Czochralskiprocess (Grovenor, C. (1989). Microelectronic Materials. CRC Press. pp.113-123). The material can present a hard, planar surface that exhibitsa uniform covering of reactive oxide (—OH) groups to a solution incontact with its surface. These oxide groups can be the attachmentpoints for subsequent silanization processes. Alternatively, alipophillic and hydrophobic surface material can be deposited thatmimics the etching characteristics of silicon oxide. Silicon nitride andsilicon carbide surfaces may also be utilized for the manufacturing ofsuitable substrates according to the various embodiments of theinvention.

In some embodiments, a passivation layer can be deposited on thesubstrate, which may or may not have reactive oxide groups. Thepassivation layer can comprise silicon nitride (Si₃N₄) or polymide. Insome instances, a photolithographic step can be used to define regionswhere the resolved loci form on the passivation layer.

The method for producing a substrate having a plurality of reactor capscan start with a substrate. The substrate (e.g., silicon) can have anynumber of layers disposed upon it, including but not limited to aconducting layer such as a metal. The conducting layer can be aluminumin some instances. In some cases, the substrate can have a protectivelayer (e.g., titanium nitride). In some cases, the substrate can have achemical layer with a high surface energy. The layers can be depositedwith the aid of various deposition techniques, such as, for example,chemical vapor deposition (CVD), atomic layer deposition (ALD), plasmaenhanced CVD (PECVD), plasma enhanced ALD (PEALD), metal organic CVD(MOCVD), hot wire CVD (HWCVD), initiated CVD (iCVD), modified CVD(MCVD), vapor axial deposition (VAD), outside vapor deposition (OVD) andphysical vapor deposition (e.g., sputter deposition, evaporativedeposition).

In some cases, an oxide layer is deposited on the substrate. In someinstances, the oxide layer can comprise silicon dioxide. The silicondioxide can be deposited using tetraethyl orthosilicate (TEOS), highdensity plasma (HDP), or any combination thereof.

In some instances, the silicon dioxide can be deposited using a lowtemperature technique. In some cases, the process is low-temperaturechemical vapor deposition of silicon oxide. The temperature is generallysufficiently low such that pre-existing metal on the chip is notdamaged. The deposition temperature can be about 50° C., about 100° C.,about 150° C., about 200° C., about 250° C., about 300° C., about 350°C., and the like. In some embodiments, the deposition temperature isbelow about 50° C., below about 100° C., below about 150° C., belowabout 200° C., below about 250° C., below about 300° C., below about350° C., and the like. The deposition can be performed at any suitablepressure. In some instances, the deposition process uses RF plasmaenergy.

In some cases, the oxide is deposited by a dry thermally grown oxideprocedure (e.g., those that may use temperatures near or exceeding1,000° C.). In some cases, the silicon oxide is produced by a wet steamprocess.

The silicon dioxide can be deposited to a thickness suitable for theformation of reactor caps that can form a plurality of resolved reactorscomprising a volume for reagents to be deposited and mixed that can besuitable for amplifying any desired amount of oligonucleotide or otherdownstream reactions as described in other paragraphs of the currentinvention.

The silicon dioxide can be deposited to any suitable thickness. In someembodiments, the silicon dioxide is about, at least about or less thanabout 1 nanometer (nm), about 2 nm, about 3 nm, about 4 nm, about 5 nm,about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 15nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm,about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm,about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm,about 300 nm, about 400 nm or about 500 nm thick.

The reactor caps can be created in a silicon dioxide substrate usingvarious manufacturing techniques that are known in the art. Suchtechniques may include semiconductor fabrication techniques. In somecases, the reactor caps are created using photolithographic techniquessuch as those used in the semiconductor industry. For example, aphoto-resist (e.g., a material that changes properties when exposed toelectromagnetic radiation) can be coated onto the silicon dioxide (e.g.,by spin coating of a wafer) to any suitable thickness. The substrateincluding the photo-resist can be exposed to an electromagneticradiation source. A mask can be used to shield radiation from portionsof the photo-resist in order to define the area of the resolved loci.The photo-resist can be a negative resist or a positive resist (e.g.,the area of the reactor caps can be exposed to electromagnetic radiationor the areas other than the reactor caps c_(a)n _(b)e exposed toelectromagnetic radiation as defined by the mask). The area overlyingthe location in which the reactor caps are to be created is exposed toelectromagnetic radiation to define a pattern that corresponds to thelocation and distribution of the reactor caps in the silicon dioxidelayer. The photoresist can be exposed to electromagnetic radiationthrough a mask defining a pattern that corresponds to the reactor caps.Next, the exposed portion of the photoresist can be removed, such as,e.g., with the aid of a washing operation (e.g., deionized water). Theremoved portion of the mask can then be exposed to a chemical etchant toetch the substrate and transfer the pattern of reactor caps into thesilicon dioxide layer. The etchant can include an acid, such as, forexample, sulfuric acid (H₂SO₄). The silicon dioxide layer can be etchedin an anisotropic fashion. Using the methods described herein, highanisotropy manufacturing methods, such as DRIE can be applied tofabricate microstructures, such as reactor caps, on or within asubstrate with side walls that deviate less than about ±3°, 2°, 1°,0.5°, 0.1°, or less from the vertical with respect to the surface of thesubstrate. Undercut values of less than about 10, 9, 8, 7, 6, 5, 4, 3,2, 1, 0.5, 0.1 μm or less can be achieved resulting in highly uniformmicrostructures.

Various etching procedures can be used to etch the silicon dioxide inthe area where the reactor caps are to be formed. The etch can be anisotropic etch (i.e., the etch rate alone one direction is equal to theetch rate along an orthogonal direction), or an anisotropic etch (i.e.,the etch rate along one direction is less than the etch rate alone anorthogonal direction), or variants thereof. The etching techniques canbe both wet silicon etches such as KOH, TMAH, EDP and the like, and dryplasma etches (for example DRIE). Both may be used to etch microstructures wafer through interconnections.

In some cases, an anisotropic etch removes the majority of the volume ofthe reactor caps. Any suitable percentage of the volume of the reactorcaps can be removed including about 60%, about 70%, about 80%, about90%, or about 95%. In some cases, at least about 60%, at least about70%, at least about 80%, at least about 90%, or at least about 95% ofthe material is removed in an anisotropic etch. In some cases, at mostabout 60%, at most about 70%, at most about 80%, at most about 90%, orat most about 95% of the material is removed in an anisotropic etch. Insome embodiments, the anisotropic etch does not remove silicon dioxidematerial all of the way through the substrate. An isotropic etch removesthe silicon dioxide material all of the way through the substratecreating a hole in some instances.

In some cases, the reactor caps are etched using a photo-lithographicstep to define the reactor caps followed by a hybrid dry-wet etch. Thephoto-lithographic step can comprise coating the silicon dioxide with aphoto-resist and exposing the photo-resist to electromagnetic radiationthrough a mask (or reticle) having a pattern that defines the reactorcaps. In some instances, the hybrid dry-wet etch comprises: (a) dryetching to remove the bulk of the silicon dioxide in the regions of thereactor caps defined in the photoresist by the photo-lithographic step;(b) cleaning the substrate; and (c) wet etching to remove the remainingsilicon dioxide from the substrate in the regions of the reactor caps.

The substrate can be cleaned with the aid of a plasma etching chemistry,or exposure to an oxidizing agent, such as, for example, H₂O₂, O₂, O₃,H₂SO₄, or a combination thereof, such as a combination of H₂O₂ andH₂SO₄. The cleaning can comprise removing residual polymer, removingmaterial that can block the wet etch, or a combination thereof. In someinstances, the cleaning is plasma cleaning. The cleaning step canproceed for any suitable period of time (e.g., 15 to 20 seconds). In anexample, the cleaning can be performed for 20 seconds with an AppliedMaterials eMAx-CT machine with settings of 100 mT, 200 W, 20 G, 20 O₂.

The dry etch can be an anisotropic etch that etches substantiallyvertically (e.g., toward the substrate) but not laterally orsubstantially laterally (e.g., parallel to the substrate). In someinstances, the dry etch comprises etching with a fluorine based etchantsuch as CF₄, CHF₃, C₂F₆, C₃F₆, or any combination thereof. In oneinstance, the etching is performed for 400 seconds with an AppliedMaterials eMax-CT machine having settings of 100 mT, 1000 W, 20 G, and50 CF4. The substrates described herein can be etched by deepreactive-ion etching (DRIE). DRIE is a highly anisotropic etchingprocess used to create deep penetration, steep-sided holes and trenchesin wafers/substrates, typically with high aspect ratios. The substratescan be etched using two main technologies for high-rate DRIE: cryogenicand Bosch. Methods of applying DRIE are described in the U.S. Pat. No.5,501,893, which is herein incorporated by reference in its entirety.

The wet etch can be an isotropic etch that removes material in alldirections. In some instances, the wet etch undercuts the photo-resist.Undercutting the photo-resist can make the photo-resist easier to removein a later step (e.g., photo-resist “lift off”). In an embodiment, thewet etch is buffered oxide etch (BOE). In some cases, the wet oxideetches are performed at room temperature with a hydrofluoric acid basethat can be buffered (e.g., with ammonium fluoride) to slow down theetch rate. Etch rate can be dependent on the film being etched andspecific concentrations of HF and/or NH₄F. The etch time needed tocompletely remove an oxide layer is typically determined empirically. Inone example, the etch is performed at 22° C. with 15:1 BOE (bufferedoxide etch).

The silicon dioxide layer can be etched up to an underlying materiallayer. For example, the silicon dioxide layer can be etched until atitanium nitride layer.

In an aspect, a method for preparing a substrate having a plurality ofreactor caps comprises etching the cavity of the reactor caps into asubstrate, such as a silicon substrate comprising a silicon dioxidelayer coated thereon using (a) a photo-lithographic step to define theresolved loci; (b) a dry etch to remove the bulk of the silicon dioxidein the regions of the reactor caps defined by the photo-lithographicstep; and (c) a wet etch to remove the remaining silicon dioxide fromthe substrate in the regions of the reactor caps. In some cases, themethod further comprises removing residual polymer, removing materialthat can block the wet etch, or a combination thereof. The method caninclude a plasma cleaning step.

In some embodiments, the photo-resist is not removed from the silicondioxide following the photo-lithographic step or the hybrid wet-dry etchin some cases. Leaving the photo-resist can be used to direct metalselectively into the reactor caps and not onto the upper surface of thesilicon dioxide layer in later steps. In some cases, the substrate iscoated with a metal (e.g., aluminum) and the wet etch does not removecertain components on the metal, e.g. those that protect the metal fromcorrosion (e.g., titanium nitride (TiN)). In some cases, however, thephotoresist layer can be removed, such as with the aid of chemicalmechanical planarization (CMP).

An exemplary nanoreactor is shown in various views in FIGS. 26A-D. Thisnanoreactor comprises 108 wells which are individually raised from abase of the nanoreactor. A cross-section of the nanoreactor is shown inFIG. 26A. A device view of the nanoreactor is shown in FIGS. 26B and26C. A handle view of the nanoreactor is shown in FIG. 26D. Ananoreactor can be configured to receive and hold liquids in a pluralityof features. The nanoreactor of FIGS. 26A-D is designed to hold liquidsin any number of the 108 wells. A nanoreactor may be contacted and/oraligned with a substrate, such as that exemplified in FIG. 25. The wellsof a nanoreactor are not limited to the configuration shown in FIG.26A-D, as any number of wells in any configuration may be arrangedwithin a nanoreactor. In some embodiments, the nanoreactor wells arearranged in a configuration which aligns with a substrate configuration.As represented by 2701, the height of a nanoreactor may be about or atleast about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8mm, 0.9 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or10 mm. In some embodiments, the height of a nanoreactor may be about orat most about 10 mm, 9.5 mm, 9 mm, 8.5 mm, 8 mm, 7.5 mm, 7 mm, 6.5 mm, 6mm, 5.5 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or0.1 mm or less. In some embodiments, the height of a nanoreactor mayrange between 0.1-10 mm, 0.2-9 mm, 0.3-8 mm, 0.4-7 mm, 0.5-6 mm, 0.6-5mm, 0.7-4 mm, 0.8-3 mm, or 0.9-2 mm. Those of skill in the artappreciate that the distance may fall within any range bound by any ofthese values, for example 0.2 mm-0.8 mm. As represented by 2702, theheight of a well of a nanoreactor may be about or at least about 0.1 mm,0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm,1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm,6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm. In someembodiments, the height of a well of a nanoreactor may be about or atmost about 10 mm, 9.5 mm, 9 mm, 8.5 mm, 8 mm, 7.5 mm, 7 mm, 6.5 mm, 6mm, 5.5 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or0.1 mm or less. In some embodiments, the height of a well of ananoreactor may range between 0.1-10 mm, 0.2-9 mm, 0.3-8 mm, 0.4-7 mm,0.5-6 mm, 0.6-5 mm, 0.7-4 mm, 0.8-3 mm, or 0.9-2 mm. Those of skill inthe art appreciate that the distance may fall within any range bound byany of these values, for example 0.1 mm-0.6 mm.

FIG. 26B includes an origin of reference indicated by a 0,0 (X,Y) axis,wherein the top left corner of an exemplary nanoreactor is diagramed. Insome embodiments, the width of the nanoreactor, represented as 2703, isfrom about 5 mm to about 150 mm along one dimension, as measured fromthe origin. In some embodiments, the width of a nanoreactor, representedas 2704, is from about 5 mm to about 150 mm along another dimension, asmeasured from the origin. In some embodiments, the width of ananoreactor in any dimension is from about 5 mm to about 125 mm, fromabout 5 mm to about 100 mm, from about 5 mm to about 75 mm, from about 5mm to about 50 mm, from about 5 mm to about 25 mm, from about 25 mm toabout 150 mm, from about 50 mm to about 150 mm, from about 75 mm toabout 150 mm, from about 100 mm to about 150 mm, or from about 125 mm toabout 150 mm. Those of skill in the art appreciate that the width mayfall within any range bound by any of these values, for example 5-25 mm.In some embodiments, the width of a nanoreactor in any dimension isabout or at least about 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm,50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140mm, or 150 mm. In some embodiments, the width of a nanoreactor in anydimension is about or at most about 150 mm, 140 mm, 130 mm, 120 mm, 110mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 50 mm, 40 mm, 30 mm, 25mm, 20 mm, 15 mm, 10 mm, or 5 mm or less.

The nanoreactor shown in FIG. 26B comprises 108 wells. The wells may bearranged in any configuration. In FIG. 26B, the wells are arranged inrows forming a square shape. Regardless of arrangement, the wells maystart at a distance of about 0.1 mm to about 149 mm from the origin, asmeasured on the X- or Y-axis and end at a distance of about 1 mm toabout 150 mm from the origin. Lengths 2706 and 2705 represent thefurthest distances of the center of a well on the X- and Y-axis from theorigin, respectively. Lengths 2710 and 2709 represent the closestdistances of the center of a well on the X- and Y-axis from the origin,respectively. In some embodiments, the furthest distance of the centerof a well in any dimension from the origin is about or at least about 1mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm,80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, or 150 mm. In someembodiments, the furthest distance of the center of a well in anydimension is about or at most about 150 mm, 140 mm, 130 mm, 120 mm, 110mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 50 mm, 40 mm, 30 mm, 25mm, 20 mm, 15 mm, 10 mm, 5 mm, 1 mm or less. In some embodiments, thefurthest distance of the center of a well in any dimension is from about5 mm to about 125 mm, from about 5 mm to about 100 mm, from about 5 mmto about 75 mm, from about 5 mm to about 50 mm, from about 5 mm to about25 mm, from about 25 mm to about 150 mm, from about 50 mm to about 150mm, from about 75 mm to about 150 mm, from about 100 mm to about 150 mm,or from about 125 mm to about 150 mm. Those of skill in the artappreciate that the distance may fall within any range bound by any ofthese values, for example 5-25 mm. In some embodiments, the closestdistance of the center of a well in any dimension from the origin isabout or at least about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm,0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, 20mm, 25 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90 mm, 100 mm, 110mm, 120 mm, 130 mm, 140 mm, or 149 mm. In some embodiments, the closestdistance of the center of a well in any dimension is about or at mostabout 149 mm, 140 mm, 130 mm, 120 mm, 110 mm, 100 mm, 90 mm, 80 mm, 70mm, 60 mm, 50 mm, 50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 5 mm,4 mm, 3 mm, 2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm,0.3 mm, 0.2 mm, 0.1 mm or less. In some embodiments, the closestdistance of the center of a well in any dimension is from about 0.1 mmto about 125 mm, from about 0.5 mm to about 100 mm, from about 0.5 mm toabout 75 mm, from about 0.5 mm to about 50 mm, from about 0.5 mm toabout 25 mm, from about 1 mm to about 50 mm, from about 1 mm to about 40mm, from about 1 mm to about 30 mm, from about 1 mm to about 20 mm, orfrom about 1 mm to about 5 mm. Those of skill in the art appreciate thatthe distance may fall within any range bound by any of these values, forexample 0.1-5 mm.

The wells of a nanoreactor may be located at any distance from the edgeof a nanoreactor. Exemplary distances between a well and an edge of ananoreactor are shown by 2707 and 2708. In some embodiments, thedistance between the center of a well and an edge of a nanoreactor inany dimension is about or at least about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm,0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, 90mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, or 149 mm. In someembodiments, the distance between the center of well and an edge of ananoreactor in any dimension is about or at most about 149 mm, 140 mm,130 mm, 120 mm, 110 mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm, 50mm, 40 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1mm or less. In some embodiments, the distance between the center of welland an edge of a nanoreactor in any dimension is from about 0.1 mm toabout 125 mm, from about 0.5 mm to about 100 mm, from about 0.5 mm toabout 75 mm, from about 0.5 mm to about 50 mm, from about 0.5 mm toabout 25 mm, from about 1 mm to about 50 mm, from about 1 mm to about 40mm, from about 1 mm to about 30 mm, from about 1 mm to about 20 mm, orfrom about 1 mm to about 5 mm. Those of skill in the art appreciate thatthe distance may fall within any range bound by any of these values, forexample 0.1-5 mm.

In some embodiments, the wells are arranged so that there exists arepeated distance between two wells. As shown by 2711 and 2712, thedistance between two wells may be from about 0.3 mm to about 9 mm apart.In some embodiments, the distance between two wells is about or at leastabout 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm 0.9 mm, 1 mm, 1.2mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3 mm,3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, 5mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6 mm, 6.8mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm, 8.6 mm,8.8 mm, or 9 mm. In some embodiments, the distance between two wells isabout or at most about 9 mm, 8.8 mm, 8.6 mm, 8.4 mm, 8.2 mm, 8 mm, 7.8mm, 7.6 mm, 7.4 mm, 7.2 mm, 7 mm, 6.8 mm, 6.6 mm, 6.4 mm, 6.2 mm, 6 mm,5.8 mm, 5.6 mm, 5.4 mm, 5.2 mm, 5 mm, 4.8 mm, 4.6 mm, 4.4 mm, 4.2 mm, 4mm, 3.8 mm, 3.6 mm, 3.4 mm, 3.2 mm, 3 mm, 2.8 mm, 2.6 mm, 2.4 mm, 2.2mm, 2 mm, 1.8 mm, 1.6 mm, 1.4 mm, 1.2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm,0.6 mm, 0.5 mm, 0.4 mm, or 0.3 mm. The distance between two wells mayrange between 0.3-9 mm, 0.4-8 mm, 0.5-7 mm, 0.6-6 mm, 0.7-5 mm, 0.7-4mm, 0.8-3 mm, or 0.9-2 mm. Those of skill in the art appreciate that thedistance may fall within any range bound by any of these values, forexample 0.8 mm-2 mm.

In some embodiments, the cross-section of the inside of a well, as shownby 2721, is about or at least about 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7mm, 0.8 mm 0.9 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm,2.4 mm, 2.6 mm, 2.8 mm, 3 mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2mm, 4.4 mm, 4.6 mm, 4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm,6.2 mm, 6.4 mm, 6.6 mm, 6.8 mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8mm, 8.2 mm, 8.4 mm, 8.6 mm, 8.8 mm, or 9 mm. In some embodiments, thecross-section of the inside of a well is about or at most about 9 mm,8.8 mm, 8.6 mm, 8.4 mm, 8.2 mm, 8 mm, 7.8 mm, 7.6 mm, 7.4 mm, 7.2 mm, 7mm, 6.8 mm, 6.6 mm, 6.4 mm, 6.2 mm, 6 mm, 5.8 mm, 5.6 mm, 5.4 mm, 5.2mm, 5 mm, 4.8 mm, 4.6 mm, 4.4 mm, 4.2 mm, 4 mm, 3.8 mm, 3.6 mm, 3.4 mm,3.2 mm, 3 mm, 2.8 mm, 2.6 mm, 2.4 mm, 2.2 mm, 2 mm, 1.8 mm, 1.6 mm, 1.4mm, 1.2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, or 0.3mm. The cross-section of the inside of a well may range between 0.3-9mm, 0.4-8 mm, 0.5-7 mm, 0.6-6 mm, 0.7-5 mm, 0.7-4 mm, 0.8-3 mm, or 0.9-2mm. Those of skill in the art appreciate that the cross-section may fallwithin any range bound by any of these values, for example 0.8 mm-2 mm.In some embodiments, the cross-section of a well, including the rim ofthe well, as shown by 2720, is about or at least about 0.3 mm, 0.4 mm,0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm 0.9 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3 mm, 3.2 mm, 3.4 mm, 3.6 mm,3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6 mm, 6.8 mm, 7 mm, 7.2 mm, 7.4 mm,7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm, 8.6 mm, 8.8 mm, or 9 mm. In someembodiments, the cross-section of a well, including the rim of the well,is about or at most about 9 mm, 8.8 mm, 8.6 mm, 8.4 mm, 8.2 mm, 8 mm,7.8 mm, 7.6 mm, 7.4 mm, 7.2 mm, 7 mm, 6.8 mm, 6.6 mm, 6.4 mm, 6.2 mm, 6mm, 5.8 mm, 5.6 mm, 5.4 mm, 5.2 mm, 5 mm, 4.8 mm, 4.6 mm, 4.4 mm, 4.2mm, 4 mm, 3.8 mm, 3.6 mm, 3.4 mm, 3.2 mm, 3 mm, 2.8 mm, 2.6 mm, 2.4 mm,2.2 mm, 2 mm, 1.8 mm, 1.6 mm, 1.4 mm, 1.2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7mm, 0.6 mm, 0.5 mm, 0.4 mm, or 0.3 mm. The cross-section of a well,including the rim of the well, may range between 0.3-9 mm, 0.4-8 mm,0.5-7 mm, 0.6-6 mm, 0.7-5 mm, 0.7-4 mm, 0.8-3 mm, or 0.9-2 mm. Those ofskill in the art appreciate that the cross-section may fall within anyrange bound by any of these values, for example 0.8 mm-2 mm.

A nanoreactor may comprise any number of wells, including but notlimited to, any number between about 2 and about 250. In someembodiments, the number of wells includes from about 2 to about 225wells, from about 2 to about 200 wells, from about 2 to about 175 wells,from about 2 to about 150 wells, from about 2 to about 125 wells, fromabout 2 to about 100 wells, from about 2 to about 75 wells, from about 2to about 50 wells, from about 2 to about 25 wells, from about 25 toabout 250 wells, from about 50 to about 250 wells, from about 75 toabout 250 wells, from about 100 to about 250 wells, from about 125 toabout 250 wells, from about 150 to about 250 wells, from about 175 toabout 250 wells, from about 200 to about 250 wells, or from about 225 toabout 250 wells. Those of skill in the art appreciate that the wellnumber may fall within any range bound by any of these values, forexample 25-125.

Fiducial marks may be placed on a nanoreactor described herein tofacilitate alignment of the nanoreactor with other components of asystem, for example a microfluidic device or a component of amicrofluidic device. Nanoreactors of the invention may have one or morefiducial marks, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fiducial marks.The device view of the nanoreactor shown in FIG. 25B comprises threefiducial marks useful for aligning the device with other components of asystem. A fiducial mark may be located at any position within thenanoreactor. As shown by 2716 and 2717, a fiducial mark may be locatednear the origin, where the fiducial mark is closer to the origin thanany one well. In some embodiments, a fiducial mark is located near anedge of the nanoreactor, as shown by 2713, where the distance from theedge is exemplified by 2714 and 2715. The fiducial mark may be locatedfrom about 0.1 mm to about 10 mm from the edge of the nanoreactor. Insome embodiments, the fiducial mark is located about or at least about0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm 0.9 mm, 1mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8mm, 3 mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm,4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6mm, 6.8 mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm,8.6 mm, 8.8 mm, 9 mm, or 10 mm from the edge of the nanoreactor. In someembodiments, the fiducial mark is located about or at most about 10 mm,9 mm, 8.8 mm, 8.6 mm, 8.4 mm, 8.2 mm, 8 mm, 7.8 mm, 7.6 mm, 7.4 mm, 7.2mm, 7 mm, 6.8 mm, 6.6 mm, 6.4 mm, 6.2 mm, 6 mm, 5.8 mm, 5.6 mm, 5.4 mm,5.2 mm, 5 mm, 4.8 mm, 4.6 mm, 4.4 mm, 4.2 mm, 4 mm, 3.8 mm, 3.6 mm, 3.4mm, 3.2 mm, 3 mm, 2.8 mm, 2.6 mm, 2.4 mm, 2.2 mm, 2 mm, 1.8 mm, 1.6 mm,1.4 mm, 1.2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm,0.3 mm, 0.2 mm, or 0.1 mm from the edge of the nanoreactor. The fiducialmark may be located between 0.1-10 mm, 0.2-9 mm, 0.3-8 mm, 0.4-7 mm,0.5-6 mm, 0.1-6 mm, 0.2-5 mm, 0.3-4 mm, 0.4-3 mm, or 0.5-2 mm from theedge of the nanoreactor. Those of skill in the art appreciate that thedistance may fall within any range bound by any of these values, forexample 0.1 mm-5 mm. The fiducial mark may be located close in distanceto a well, where exemplary X- and Y-axis distances are indicated by 2719and 2718, respectively. In some embodiments, a distance between a welland a fiducial mark is about or at least about 0.001 mm, 0.005 mm, 0.01mm, 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9mm, 1 mm, 1.2 mm, 1.5 mm, 1.7 mm, 2 mm, 2.2 mm, 2.5 mm, 2.7 mm, 3 mm,3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, or 8 mm. In someembodiments, a distance between a well and a fiducial mark is about orat most about 8 mm, 6.5 mm, 6 mm, 5.5 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3mm, 2.7 mm, 2.5 mm, 2.2 mm, 2 mm, 1.7 mm, 1.5 mm, 1.2 mm, 1 mm, 0.9 mm,0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.09 mm,0.08 mm, 0.07 mm, 0.06 mm, 0.05 mm, 0.04 mm, 0.03 mm, 0.02 mm, 0.01 mm,0.005 mm, or 0.001 mm. The distance between a well and a fiducial markmay be in a range between 0.001-8 mm, 0.01-7 mm, 0.05-6 mm, 0.1-5 mm,0.5-4 mm, 0.6-3 mm, 0.7-2 mm, or 0.8-1.7 mm. Those of skill in the artappreciate that the distance may fall within any range bound by any ofthese values, for example 0.5-2 mm.

The handle view of the nanoreactor shown in FIG. 26D comprises fourfiducial marks useful for aligning the device with other components of asystem. A fiducial mark may be located at any position within thenanoreactor. As shown by 2722 and 2723 on the detailed view of thefiducial mark H, a fiducial mark may be located near a corner of ananoreactor on the handle side. The fiducial mark may be located fromabout 0.1 mm to about 10 mm from the corner of the nanoreactor. In someembodiments, the fiducial mark is located about or at least about 0.1mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm 0.9 mm, 1 mm,1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6 mm,6.8 mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm, 8.6mm, 8.8 mm, 9 mm, or 10 mm from the corner of the nanoreactor. In someembodiments, the fiducial mark is located about or at most about 10 mm,9 mm, 8.8 mm, 8.6 mm, 8.4 mm, 8.2 mm, 8 mm, 7.8 mm, 7.6 mm, 7.4 mm, 7.2mm, 7 mm, 6.8 mm, 6.6 mm, 6.4 mm, 6.2 mm, 6 mm, 5.8 mm, 5.6 mm, 5.4 mm,5.2 mm, 5 mm, 4.8 mm, 4.6 mm, 4.4 mm, 4.2 mm, 4 mm, 3.8 mm, 3.6 mm, 3.4mm, 3.2 mm, 3 mm, 2.8 mm, 2.6 mm, 2.4 mm, 2.2 mm, 2 mm, 1.8 mm, 1.6 mm,1.4 mm, 1.2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm,0.3 mm, 0.2 mm, or 0.1 mm from the corner of the nanoreactor. Thefiducial mark may be located between 0.1-10 mm, 0.2-9 mm, 0.3-8 mm,0.4-7 mm, 0.5-6 mm, 0.1-6 mm, 0.2-5 mm, 0.3-4 mm, 0.4-3 mm, or 0.5-2 mmfrom the corner of the nanoreactor. Those of skill in the art appreciatethat the distance may fall within any range bound by any of thesevalues, for example 0.1 mm-5 mm. The fiducial mark may have any widthsuitable for function. In some embodiments, as exemplified by 2724 and2725, the width of a fiducial mark is about or at least about 0.1 mm,0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm 0.9 mm, 1 mm, 1.2mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3 mm,3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, 5mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6 mm, 6.8mm, 7 mm, 7.2 mm, 7.4 mm, 7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm, 8.6 mm,8.8 mm, 9 mm, or 10 mm. In some embodiments, the width of a fiducialmark is about or at most about 10 mm, 9 mm, 8.8 mm, 8.6 mm, 84 mm, 8.2mm, 8 mm, 7.8 mm, 7.6 mm, 7.4 mm, 7.2 mm, 7 mm, 6.8 mm, 6.6 mm, 6.4 mm,6.2 mm, 6 mm, 5.8 mm, 5.6 mm, 5.4 mm, 5.2 mm, 5 mm, 4.8 mm, 4.6 mm, 4.4mm, 4.2 mm, 4 mm, 3.8 mm, 3.6 mm, 3.4 mm, 3.2 mm, 3 mm, 2.8 mm, 2.6 mm,2.4 mm, 2.2 mm, 2 mm, 1.8 mm, 1.6 mm, 1.4 mm, 1.2 mm, 1 mm, 0.9 mm, 0.8mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm. Thefiducial mark width may range between 0.1-10 mm, 0.2-9 mm, 0.3-8 mm,0.4-7 mm, 0.5-6 mm, 0.1-6 mm, 0.2-5 mm, 0.3-4 mm, 0.4-3 mm, or 0.5-2 mmlong. Those of skill in the art appreciate that the width may fallwithin any range bound by any of these values, for example 0.1 mm-5 mm.A cross-section of a fiducial mark may be of any suitable size, as shownin by 2726. In some embodiments, the cross-section of a fiducial mark isabout or at least about 0.001 mm, 0.002 mm, 0.004 mm, 0.006 mm, 0.008mm, 0.01 mm, 0.012 mm, 0.014 mm, 0.016 mm, 0.018 mm, 0.02 mm, 0.025 mm,0.03 mm, 0.035 mm, 0.04 mm, 0.045 mm, 0.05 mm, 0.055 mm, 0.06 mm, 0.065mm, 0.07 mm, 0.075 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, or 0.5mm. In some embodiments, the cross-section of a fiducial mark is aboutor at most about 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.08 mm, 0.075mm, 0.07 mm, 0.065 mm, 0.06 mm, 0.055 mm, 0.05 mm, 0.045 mm, 0.04 mm,0.035 mm, 0.03 mm, 0.025 mm, 0.02 mm, 0.018 mm, 0.016 mm, 0.014 mm,0.012 mm, 0.01 mm, 0.008 mm, 0.006 mm, 0.004 mm, 0.002 mm, 0.001 mm orless. The cross-section of a fiducial mark may range between 0.001-0.5mm, 0.004-0.4 mm, 0.008-0.3 mm, 0.01-0.2 mm, 0.015-0.1 mm, 0.018-0.1 mm,or 0.02-0.05 mm. Those of skill in the art appreciate that thecross-section may fall within any range bound by any of these values,for example 0.02 mm-0.1 mm.

In some embodiments, the nanoreactor may have a location for a label ora serial label, as exemplified in FIG. 26E depicting an exemplary layoutof wells in a nanoreactor. In some embodiments, the label is a serialnumber. The label may be located near an edge of the nanoreactor, asexemplified by the distances 2728 and 2727. In some embodiments, anyportion of the label is located from about 0.1 mm to about 10 mm fromthe edge of the nanoreactor. In some embodiments, any portion of thelabel is located about or at least about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm,0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm 0.9 mm, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3 mm, 3.2 mm, 3.4 mm, 3.6 mm,3.8 mm, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6mm, 5.8 mm, 6 mm, 6.2 mm, 6.4 mm, 6.6 mm, 6.8 mm, 7 mm, 7.2 mm, 7.4 mm,7.6 mm, 7.8 mm, 8 mm, 8.2 mm, 8.4 mm, 8.6 mm, 8.8 mm, 9 mm, or 10 mmfrom the edge of a nanoreactor. In some embodiments, the any portion ofthe label is located about or at most about 10 mm, 9 mm, 8.8 mm, 8.6 mm,8.4 mm, 8.2 mm, 8 mm, 7.8 mm, 7.6 mm, 7.4 mm, 7.2 mm, 7 mm, 6.8 mm, 6.6mm, 6.4 mm, 6.2 mm, 6 mm, 5.8 mm, 5.6 mm, 5.4 mm, 5.2 mm, 5 mm, 4.8 mm,4.6 mm, 4.4 mm, 4.2 mm, 4 mm, 3.8 mm, 3.6 mm, 3.4 mm, 3.2 mm, 3 mm, 2.8mm, 2.6 mm, 2.4 mm, 2.2 mm, 2 mm, 1.8 mm, 1.6 mm, 1.4 mm, 1.2 mm, 1 mm,0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1mm from the edge of a nanoreactor. The distance may be in a rangebetween 0.1-10 mm, 0.2-9 mm, 0.3-8 mm, 0.4-7 mm, 0.5-6 mm, 0.6-5 mm,0.7-4 mm, 0.8-3 mm, 0.9-2 mm or 1.5 mm. Those of skill in the artappreciate that the distance may fall within any range bound by any ofthese values, for example 0.5-2 mm. The label may have any length,including from about 1 mm to about 25 mm as exemplified by 2726. In someembodiments, the length of a label is about or at least about 1 mm, 5mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80mm, 90 mm, 100 mm, 110 mm, 120 mm, 130 mm, 140 mm, or 150 mm. In someembodiments, the length of a label is about or at most about 150 mm, 140mm, 130 mm, 120 mm, 110 mm, 100 mm, 90 mm, 80 mm, 70 mm, 60 mm, 50 mm,50 mm, 40 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 5 mm, 1 mm or less. Insome embodiments, the length of a label is from about 5 mm to about 125mm, from about 5 mm to about 100 mm, from about 5 mm to about 75 mm,from about 5 mm to about 50 mm, from about 5 mm to about 25 mm, fromabout 25 mm to about 150 mm, from about 50 mm to about 150 mm, fromabout 75 mm to about 150 mm, from about 100 mm to about 150 mm, or fromabout 125 mm to about 150 mm. Those of skill in the art appreciate thatthe length may fall within any range bound by any of these values, forexample 5-25 mm.

Materials

The substrates, the solid support or the microstructures or reactorstherein may be fabricated from a variety of materials, suitable for themethods and compositions of the invention described herein. In certainembodiments, the materials from which the substrates/solid supports ofthe comprising the invention are fabricated exhibit a low level ofoligonucleotide binding. In some situations, material that aretransparent to visible and/or UV light can be employed. Materials thatare sufficiently conductive, e.g. those that can form uniform electricfields across all or a portion of the substrates/solids supportdescribed herein, can be utilized. In some embodiments, such materialsmay be connected to an electric ground. In some cases, the substrate orsolid support can be heat conductive or insulated. The materials can bechemical resistant and heat resistant to support chemical or biochemicalreactions such as a series of oligonucleotide synthesis reaction. Forflexible materials, materials of interest can include: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like.For rigid materials, specific materials of interest include: glass; fusesilica; silicon, plastics (for example polytetraflouroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). Thesubstrate, solid support or reactors can be fabricated from a materialselected from the group consisting of silicon, polystyrene, agarose,dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane(PDMS), and glass. The substrates/solid supports or the microstructures,reactors therein may be manufactured with a combination of materialslisted herein or any other suitable material known in the art.

Surface Modifications

In various embodiments, surface modifications are employed for thechemical and/or physical alteration of a surface by an additive orsubtractive process to change one or more chemical and/or physicalproperties of a substrate surface or a selected site or region of asubstrate surface. For example, surface modification may involve (1)changing the wetting properties of a surface, (2) functionalizing asurface, i.e., providing, modifying or substituting surface functionalgroups, (3) defunctionalizing a surface, i.e., removing surfacefunctional groups, (4) otherwise altering the chemical composition of asurface, e.g., through etching, (5) increasing or decreasing surfaceroughness, (6) providing a coating on a surface, e.g., a coating thatexhibits wetting properties that are different from the wettingproperties of the surface, and/or (7) depositing particulates on asurface.

The substrate surface, or the resolved loci, onto which theoligonucleotides or other moieties are deposited may be smooth orsubstantially planar, or have irregularities, such as depressions orelevations. The surface may be modified with one or more differentlayers of compounds that serve to modify the properties of the surfacein a desirable manner. Such modification layers of interest include:inorganic and organic layers such as metals, metal oxides, polymers,small organic molecules and the like. Polymeric layers of interestinclude layers of: peptides, proteins, nucleic acids or mimetics thereof(for example, peptide nucleic acids and the like); polysaccharides,phospholipids, polyurethanes, polyesters, polycarbonates, polyureas,polyamides, polyetheyleneamines, polyarylene sulfides, polysiloxanes,polyimides, polyacetates, and the like, or any other suitable compoundsdescribed herein or otherwise known in the art, where the polymers maybe hetero- or homopolymeric, and may or may not have separate functionalmoieties attached thereto (for example, conjugated). Other materials andmethods for surface modification of the substrate or coating of thesolid support are described in U.S. Pat. No. 6,773,888 and U.S. Pub. No.2007/0054127, which are herein incorporated by reference in theirentirety.

The resolved loci can be functionalized with a moiety that can increaseor decrease the surface energy of the solid support. The moiety can bechemically inert or alternatively, be a moiety that is suited to supporta desired chemical reaction. The surface energy, or hydrophobicity, of asurface can determine the affinity of an oligonucleotide to attach ontothe surface. A method for preparing a substrate can comprise: (a)providing a substrate having a surface that comprises silicon dioxide;and (b) silanizing the surface using, a suitable silanizing agentdescribed herein or otherwise known in the art, for example, anorganofunctional alkoxysilane molecule. In some cases, theorganofunctional alkoxysilane molecule can bedimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane,trichloro-octodecyl-silane, trimethyl-octodecyl-silane,triethyl-octodecyl-silane or any combination thereof.

The surface of the substrate can also be prepared to have a low surfaceenergy using any method that is known in the art. Lowering the surfaceenergy can facilitate oligonucleotides to attach to the surface. Thesurface can be functionalized to enable covalent binding of molecularmoieties that can lower the surface energy so that wettability can bereduced. In some embodiments, the functionalization of surfaces enablesan increase in surface energy and wettability.

In some embodiments, the surface of the substrate is contacted with aderivatizing composition that contains a mixture of silanes, underreaction conditions effective to couple the silanes to the substratesurface, typically via reactive hydrophilic moieties present on thesubstrate surface. Silanization generally can be used to cover a surfacethrough self-assembly with organofunctional alkoxysilane molecules. Avariety of siloxane functionalizing reagents can further be used ascurrently known in the art, e.g. for lowering or increasing surfaceenergy. The organofunctional alkoxysilanes are classified according totheir organic functions. Non-limiting examples of siloxanefunctionalizing reagents include hydroxyalkyl siloxanes (silylatesurface, functionalizing with diborane and oxidizing the alcohol byhydrogen peroxide), diol (dihydroxyalkyl) siloxanes (silylate surface,and hydrolyzing to diol), aminoalkyl siloxanes (amines require nointermediate functionalizing step), glycidoxysilanes(3-glycidoxypropyl-dimethyl-ethoxysilane, glycidoxy-trimethoxysilane),mercaptosilanes (3-mercaptopropyl-trimethoxysilane, 3-4epoxycyclohexyl-ethyltrimethoxysilane or3-mercaptopropyl-methyl-dimethoxysilane),bicyclohepthenyl-trichlorosilane, butyl-aldehydr-trimethoxysilane, ordimeric secondary aminoalkyl siloxanes. The hydroxyalkyl siloxanes caninclude allyl trichlorochlorosilane turning into 3-hydroxypropyl, or7-oct-1-enyl trichlorochlorosilane turning into 8-hydroxyoctyl. The diol(dihydroxyalkyl) siloxanes include glycidyl trimethoxysilane-derived(2,3-dihydroxypropyloxy)propyl. The aminoalkyl siloxanes include3-aminopropyl trimethoxysilane turning into 3-aminopropyl(3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane,3-aminopropyl-dimethyl-ethoxysilane, or 3-aminopropyl-trimethoxysilane).The dimeric secondary aminoalkyl siloxanes can be bis(3-trimethoxysilylpropyl) amine turning into bis(silyloxylpropyl)amine.In addition, a number of alternative functionalized surfaces can be usedin the present invention. Non-limiting examples include thefollowing: 1. polyethylene/polypropylene (functionalized by gammairradiation or chromic acid oxidation, and reduction to hydroxyalkylsurface); 2. highly crosslinked polystyrene-divinylbenzene (derivatizedby chloromethylation, and aminated to benzylamine functional surface);3. nylon (the terminal aminohexyl groups are directly reactive); or 4.etched, reduced polytetrafluoroethylene. Other methods andfunctionalizing agents are described in U.S. Pat. No. 5,474,796, whichis herein incorporated by reference in its entirety. The mixture offunctionalization groups, e.g. silanes, can be in any different ratios.For example, without limitation, the mixture can comprise at least twodifferent types of functionalization agents, e.g. silanes. The ratio ofthe at least two types of surface functionalization agents, e.g.silanes, in a mixture can be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:5,3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:16, 3:17, 3:19, 4:5, 4:7, 4:9, 4:11,4:13, 4:15, 4:17, 4:19, 5:6, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16,5:17, 5:18, 5:19, 6:7, 6:11, 6:13, 6:17, 6:19, 7:8, 7:9, 7:10, 7:11,7:12, 7:13, 7:15, 7:16, 7:18, 7:19, 8:9, 8:11, 8:13, 8:15, 8:17, 8:19,9:10, 9:11, 9:13, 9:14, 9:16, 9:17, 9:19, 10:11, 10:13, 10:17, 10:19,11:12, 11:13, 11:14, 11:15, 11:16, 11:17, 11:18, 11:19, 11:20, 12:13,12:17, 12:19, 13:14, 13:15, 13:16, 13:17, 13:18, 13:19, 13:20, 14:15,14:17, 14:19, 15:16, 15:17, 15:19, 16:17, 16:19, 17:18, 17:19, 17:20,18:19, 19:20, or any other ratio to achieve a desired surfacerepresentation of two groups. Without being bound by theory, it isunderstood that surface representation will be highly proportional tothe ration of two groups in a mixture. Desired surface tensions,wettabilities, water contact angles, or contact angles for othersuitable solvents according to the methods and compositions of theinvention can be achieved by providing a ratio of functionalizationagents. Further, the agents in the mixture maybe chosen from suitablereactive and inert moieties for downstream reactions, diluting thesurface density of reactive groups to a desired level according to themethods and compositions of the invention. In some embodiments, thedensity of the fraction of a surface functional group that reacts toform a growing oligonucleotide in an oligonucleotide synthesis reactionis about, less than about, or greater than about 0.005, 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5,5.0, 7.0, 10.0, 15.0, 20.0, 50.0, 75.0, 100.0 μMol/m².

In various embodiments, the surface is modified to have a higher surfaceenergy, or become more hydrophilic with a coating of reactivehydrophilic moieties. By altering the surface energy of different partsof the substrate surface, the spreading of the deposited reagent liquidcan be adjusted, in some cases facilitated. For example, FIG. 5A-Cillustrates a case when a droplet of reagent is deposited into amicrowell by an inkjet printer. The liquid droplet can spread over andfill the smaller microwells because the surface of the microwells hashigher surface energy compared to the other surface nearby in this case.The reactive hydrophilic moieties on the substrate surface can behydroxyl groups, carboxyl groups, thiol groups, and/or substituted orunsubstituted amino groups. Suitable materials include, but are notlimited to, supports that can be used for solid phase chemicalsynthesis, e.g., cross-linked polymeric materials (e.g., divinylbenzenestyrene-based polymers), agarose (e.g., Sepharose®), dextran (e.g.,Sephadex®), cellulosic polymers, polyacrylamides, silica, glass(particularly controlled pore glass, or “CPG”), ceramics, and the like.The supports may be obtained commercially and used as is, or they may betreated or coated prior to functionalization.

Hydrophilic and Hydrophobic Surfaces

The surface energy, or hydrophobicity of a surface, can be evaluated ormeasured by measuring a water contact angle. Water contact angle is theangle between the drop surface and a solid surface where a water dropletmeets the solid surface. The solid surface can be a smooth, flat orplanar surface. It can quantify the wetting of a solid surface by aliquid (e.g., water) via the Young equation. In some cases, watercontact angle hysteresis can be observed, ranging from the so-calledadvancing (maximal) water contact angle to the receding (minimal) watercontact angle. The equilibrium water contact can be found within thosevalues, and can be calculated from them. Hydrophobicity andhydrophilicity can be expressed in relative quantitative terms usingwater contact angle. A surface with a water contact angle of smallerthan 90°, the solid surface can be considered hydrophilic or polar. Asurface with a water contact angle of greater than 90°, the solidsurface can be considered hydrophobic or apolar. Highly hydrophobicsurfaces with low surface energy can have water contact angle that isgreater than 120°.

Surface characteristics of coated surfaces can be adjusted in variousways suitable for oligonucleotide synthesis. The surface can be selectedto be inert to the conditions of ordinary oligonucleotide synthesis;e.g. the solid surface may be devoid of free hydroxy, amino, or carboxylgroups to the bulk solvent interface during monomer addition, dependingon the selected chemistry. Alternatively, the surface may comprisereactive moieties prior to the start of the first cycle, or first fewcycles of the oligonucleotide synthesis and these reactive moieties canbe quickly depleted to unmeasurable densities after one, two, three,four, five, or more cycles of the oligonucleotide synthesis reaction.The surface can further be optimized for well or poor wetting, e.g. bycommon organic solvents such as acetonitrile and the glycol ethers oraqueous solvents, relative to surrounding surfaces.

Without being bound by theory, the wetting phenomenon is understood tobe a measure of the surface tension or attractive forces betweenmolecules at a solid-liquid interface, and is expressed in dynes/cm2.For example, fluorocarbons have very low surface tension, which istypically attributed to the unique polarity (electronegativity) of thecarbon-flourine bond. In tightly structured Langmuir-Blodgett typefilms, surface tension of a layer can be primarily determined by thepercent of fluorine in the terminus of the alkyl chains. For tightlyordered films, a single terminal trifluoromethyl group can render asurface nearly as lipophobic as a perfluoroalkyl layer. Whenfluorocarbons are covalently attached to an underlying derivatized solid(e.g. a highly crosslinked polymeric) support, the density of reactivesites can be lower than Langmuir-Blodgett and group density. Forexample, surface tension of a methyltrimethoxysilane surface can beabout 22.5 mN/m and aminopropyltriethoxysilane surface can be about 35mN/m. Other examples of silane surfaces are described in Arkles B etal., “The role of polarity in the structure of silanes employed insurface modification”, Silanes and Other Coupling Agents, Vol. 5, whichis herein incorporated by reference in its entirety. Briefly,hydrophilic behavior of surfaces is generally considered to occur whencritical surface tensions are greater than 45 mN/m. As the criticalsurface tension increases, the expected decrease in contact angle isaccompanied with stronger adsorptive behavior. Hydrophobic behavior ofsurfaces is generally considered to occur when critical surface tensionsare less than 35 mN/m. At first, the decrease in critical surfacetension is associated with oleophilic behavior, i.e. the wetting of thesurfaces by hydrocarbon oils. As the critical surface tensions decreasebelow 20 mN/m, the surfaces resist wetting by hydrocarbon oils and areconsidered both oleophobic as well as hydrophobic. For example, silanesurface modification can be used to generate a broad range of criticalsurface tensions. Accordingly, the methods and compositions of theinvention may use surface coatings, e.g. those involving silanes, toachieve surface tensions of less than 5, 6, 7, 8, 9, 10, 12, 15, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 115, 120 mN/m, or higher.Further, the methods and compositions of the invention may use surfacecoatings, e.g. those involving silanes, to achieve surface tensions ofmore than 115, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15,12, 10, 9, 8, 7, 6 mN/m or less. The water contact angle and the surfacetension of non-limiting examples of surface coatings, e.g., thoseinvolving silanes, are described in Table 1 and Table 2 of Arkles et al.(Silanes and Other Coupling Agents, Vol. 5v: The Role of Polarity in theStructure of Silanes Employed in Surface Modification. 2009), which isincorporated herein by reference in its entirety. The tables arereplicated below.

TABLE 1 Contact angles of water (degrees) on smooth surfacesHeptadecafluorodecyltrimethoxysilane 113-115 Poly(tetrafluoroethylene)108-112 Polypropylene 108 Octadecyldimethylchlorosilane 110Octadecyltrichlorosilane 102-109Tris(trimethylsiloxy)silylethyldimethylchlorosilane 103-104Octyldimethylchlorosilane 104 Butyldimethylchlorosilane 100Trimethylchlorosilane  90-100 Polyethylene  88-103 Polystyrene 94Poly(chlorotrifluoroethylene) 90 Human skin 75-90 Diamond 87 Graphite 86Silicon (etched) 86-88 Talc 82-90 Chitosan 80-81 Steel 70-75Methoxyethoxyundecyltrichlorosilane 73-74Methacryloxypropyltrimethoxysilane 70 Gold, typical (see gold, clean) 66Intestinal mucosa 50-60 Kaolin 42-46 Platinum 40 Silicon nitride 28-30Silver iodide 17 [Methoxy(polyethyleneoxy)propyl]trimethoxysilane 15-16Sodalime glass <15 Gold, clean <10 Trimethoxysilylpropyl substitutedpoly(ethyleneimine), <10 hydrochloride Note: In Table 1, contact anglesfor silanes refer to hydrolytic deposition of the silane onto smoothsurfaces. The data here are drawn from various literature sources andfrom the authors' work. Exact comparisons between substrates do not takeinto account differences in test methods or whether advancing, recedingor equilibrium contact angles were reported.

TABLE 2 Critical surface tensions (mN/m)Heptadecafluorodecyltrichlorosilane 12 Poly(tetrafluoroethylene) 18.5Octadecyltrichlorosilane 20-24 Methyltrimethoxysilane 22.5Nonafluorohexyltrimethoxysilane 23 Vinyltriethoxysilane 25 Paraffin wax25.5 Ethyltrimethoxysilane 27.0 Propyltrimethoxysilane 28.5 Glass,sodalime (wet) 30.0 Poly(chlorotrifluoroethylene) 31.0 Polypropylene31.0 Poly(propylene oxide) 32 Polyethylene 33.0Trifluoropropyltrimethoxysilane 33.53-(2-Aminoethyl)aminopropyltrimethoxysilane 33.5 Polystyrene 34p-Tolyltrimethoxysilane 34 Cyanoethyltrimethoxysilane 34Aminopropyltriethoxysilane 35 Acetoxypropyltrimethoxysilane 37.5Poly(methyl methacrylate) 39 Poly(vinyl chloride) 39Phenyltrimethoxysilane 40.0 Chloropropyltrimethoxysilane 40.5Mercaptopropyltrimethoxysilane 41 Glycidoxypropyltrimethoxysilane 42.5Poly(ethylene terephthalate) 43 Copper (dry) 44 Poly(ethylene oxide)43-45 Aluminum (dry) 45 Nylon 6/6 45-46 Iron (dry) 46 Glass, sodalime(dry) 47 Titanium oxide (anatase) 91 Ferric oxide 107 Tin oxide 111

Methods to measure water contact angle can use any method that is knownin the art, including the static sessile drop method, the dynamicsessile drop method, dynamic Wilhelmy method, single-fiber Wilhelmymethod, powder contact angle method, and the like. In some cases, thesurface of the substrate, or a portion of the surface of the substrateas described herein in the current invention can be functionalized ormodified to be hydrophobic, to have a low surface energy, or to have awater contact angle that would be measured to be greater than about 90°,95°, 100°, 105°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145° or 150°on an uncurved, smooth, or planar equivalent of the relevantfunctionalized surface of the substrate, as described herein. The watercontact angle of a functionalized surface described herein can refer tothe contact angle of a water droplet on the functionalized surface in anuncurved, smooth, flat and planar geometry. In some cases, the surfaceof the substrate, or a portion of the surface of the substrate asdescribed herein in the current invention can be functionalized ormodified to be hydrophilic, to have a high surface energy, or to have awater contact angle that would be measured to be less than about 90°,85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°,15° or 10° on an uncurved, smooth or planar equivalent of the relevantfunctionalized surface of the substrate, as described herein. Thesurface of the substrate or a portion of the surface of the substratecan be functionalized or modified to be more hydrophilic or hydrophobicas compared to the surface or the portion of the surface prior to thefunctionalization or modification.

In some cases, one or more surfaces can be modified to have a differencein water contact angle of greater than 90°, 85°, 80°, 75°, 70°, 65°,60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured onone or more uncurved, smooth or planar equivalent surfaces. In somecases, the surface of the microstructures, channels, resolved loci,resolved reactor caps or other parts of the substrate may be modified tohave a differential hydrophobicity corresponding to a difference inwater contact angle that is greater than 90°, 85°, 80°, 75°, 70°, 65°,60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15° or 10° as measured onuncurved, smooth or planar equivalent surfaces of such structures.Unless otherwise stated, water contact angles mentioned hereincorrespond to measurements that would be taken on uncurved, smooth orplanar equivalents of the surfaces in question.

Other methods for functionalizing the surface are described in U.S. Pat.No. 6,028,189, which is herein incorporated by reference in itsentirety. For example, hydrophilic resolved loci can be generated byfirst applying a protectant, or resist, over each loci within thesubstrate. The unprotected area can be then coated with a hydrophobicagent to yield an unreactive surface. For example, a hydrophobic coatingcan be created by chemical vapor deposition of(tridecafluorotetrahydrooctyl)-triethoxysilane onto the exposed oxidesurrounding the protected circles. Finally, the protectant, or resist,can be removed exposing the loci regions of the substrate for furthermodification and oligonucleotide synthesis. In some embodiments, theinitial modification of such unprotected regions may resist furthermodification and retain their surface functionalization, while newlyunprotected areas can be subjected to subsequent modification steps.

Multiple Parallel Microfluidic Reactions

In another aspect, systems and methods for conducting a set of parallelreactions are described herein. The system may comprise two or moresubstrates that can be sealed, e.g. releasably sealed, with each other,forming a plurality of individually addressable reaction volumes orreactors upon sealing. New sets of reactors may be formed by releasing afirst substrate from a second substrate and aligning it with a thirdsubstrate. Each substrate can carry reagents, e.g. oligonucleotides,enzymes, buffers, or solvents, for desired reactions. In someembodiments, the system comprises a first surface with a plurality ofresolved loci at a first suitable density and a capping element with aplurality of resolved reactor caps at a second suitable density. Thesystem can align the plurality of resolved reactor caps with theplurality of resolved loci on the first surface forming a temporary sealbetween the first surface and the capping element. The temporary sealbetween the aligned substrates may physically divide the loci on thefirst surface into groups of about at least about, or less than about 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200loci, or more. A set of parallel reactions described herein can beconducted according to the methods and compositions of the invention. Afirst surface with a plurality of resolved loci at a first density and acapping element with a plurality of resolved reactor caps at a seconddensity can be aligned, such that the plurality of resolved reactor capswith the plurality of resolved loci on the first surface form atemporary seal between the first surface and the capping element andthereby physically divide the loci on the first surface into groups ofabout at least about, or less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 125, 150, 200 loci, or more. A firstreaction can be performed, forming a first set of reagents. The cappingelement may be released from the first surface. Upon release, thereactor caps may each retain at least a portion of the first set ofreagents in the previously sealed reaction volumes. The plurality ofresolved loci can be at a density of about, at least about or less thanabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 15, about 20, about 25, about 30, about 35,about 40, about 50, about 75, about 100, about 200, about 300, about400, about 500, about 600, about 700, about 800, about 900, about 1000,about 1500, about 2000, about 3000, about 4000, about 5000, about 6000,about 7000, about 8000, about 9000, about 10000, about 20000, about40000, about 60000, about 80000, about 100000, or about 500000 per 1mm². In some embodiments, the plurality of resolved loci can be at adensity of about, at least about, less than about 100 per mm². Theplurality of resolved reactor caps can be at a density of about, atleast about, less than about 1 per mm². In some embodiments, theplurality of resolved reactor caps can be at a density of about, atleast about or less than about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 15, about 20, about 25, about30, about 35, about 40, about 50, about 75, about 100, about 200, about300, about 400, about 500, about 600, about 700, about 800, about 900,about 1000, about 1500, about 2000, about 3000, about 4000, about 5000,about 6000, about 7000, about 8000, about 9000, about 10000, about20000, about 40000, about 60000, about 80000, about 100000, or about500000 per 1 mm². The methods described herein can further compriseproviding a second surface with a plurality of resolved loci at a thirddensity and aligning the plurality of resolved reactor caps with theplurality of resolved loci on the second surface. and forming a seal,typically a temporary or releasable seal, between the second surface andthe capping element. The newly formed sealed may physically divide theloci on the second surface into groups of about at least about, or lessthan about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,125, 150, 200 loci, or more. A second reaction may be performed,optionally using a portion of the first set of reagents, thereby forminga second set of reagents. The capping element may be released from thesecond surface. Upon release, the reactor caps may each retain at leasta portion of the second set of reagents in the previously sealed secondreaction volumes. In some cases, the second surface with a plurality ofresolved loci can have a locus density of at least about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 15, about 20, about 25, about 30, about 35, about 40, about 50,about 75, about 100, about 200, about 300, about 400, about 500, about600, about 700, about 800, about 900, about 1000, about 1500, about2000, about 3000, about 4000, about 5000, about 6000, about 7000, about8000, about 9000, about 10000, about 20000, about 40000, about 60000,about 80000, about 100000, or about 500000 per 1 mm². Various aspects ofthe embodiments of the systems, methods and instrumentations aredescribed herein.

The system assembly can comprise any number of static wafers and anynumber of dynamic wafers. For example, the system can comprise threesubstrates in a column and four substrates in a row. The transportsystem can comprise three static wafers (or substrates) and one dynamicwafer (or substrate). The dynamic wafers can move or transport inbetween a plurality of static wafers. A dynamic wafer can be transportedbetween three statically mounted wafers. In some embodiments, thedynamic wafer can have a diameter that is about 50, 100, 150, 200 or 250mm or 2, 4, 6, or 8 in or higher. The dynamic wafers can be mounted in atemperature controlled vacuum chuck. The systems of the invention allowfor configurations, wherein the dynamic wafers can move in Z direction,which may be the direction that is perpendicular to the surface of awafer that is to face a surface of a second wafer, with about or lessthan about 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5 or 3 μm control ofz-position, and can align theta_z of wafers, the angle between thenormals of the surfaces of two wafers that are to face each other, e.g.by matching a pattern on the dynamic wafer with another pattern on thestatic wafer within a range of tolerance. The wafer positioningtolerances can be about or less than about 1, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 150, 200, 250, 300 350, 400, 450 or 500 micro radiansin difference in angle of rotation in x-y plane. In some embodiments,the wafer positioning tolerances can be about or less than about 50micro radians in difference in angle of rotation in x-y plane. The waferpositioning tolerances can be about or less than about 0.01, 0.05, 0.1,0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μm of distancein x-direction. The wafer positioning tolerances can be about or lessthan about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 μm of distance in y-direction. The wafer positioningtolerances can be about or less than about 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 micro radians in rotations of x-y plane in z-direction. In someembodiments, the wafer positioning tolerances can be about or less thanabout 5 micro radians in rotations of x-y plane in z-direction. In someembodiments, the wafer positioning tolerances can be about or less thanabout 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 μm ofdistance in z-direction. In some embodiments, the wafer positioningtolerances can be about or less than about 0.5 μm of distance inz-direction.

In some cases, the systems and methods for conducting a set of parallelreactions can further comprise a third, a four, a fifth, a sixth, aseventh, a eighth, a ninth or a tenth surface with a plurality ofresolved loci and/or a capping element with a plurality of resolvedreactor caps. The third, the four, the fifth, the sixth, the seventh,the eighth, the ninth or the tenth surfaces can be aligned and can forma temporary seal between the two surfaces and the corresponding cappingelement, thereby physically dividing the loci and/or reactor caps on thesurfaces. A third, a four, a fifth, a sixth, a seventh, a eighth, aninth or a tenth reaction can be performed using a portion of thereagents that is retained from the previous reaction, namely, thesecond, a third, a four, a fifth, a sixth, a seventh, a eighth or aninth set of reagents, thereby forming the third, the four, the fifth,the sixth, the seventh, the eighth, the ninth or the tenth set ofreagents. Each of the capping elements described herein can be releasedfrom its corresponding surface, wherein the reactor caps can retain atleast a portion of the previous set of reagents of another reactionvolume. In some cases, the second surface with a plurality of resolvedloci can be at a density of at least 2/mm². In some embodiments, thesecond surface with a plurality of resolved loci can have a locusdensity of at least about 1, about 2, about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10, about 15, about 20, about 25,about 30, about 35, about 40, about 50, about 75, about 100, about 200,about 300, about 400, about 500, about 600, about 700, about 800, about900, about 1000, about 1500, about 2000, about 3000, about 4000, about5000, about 6000, about 7000, about 8000, about 9000, about 10000, about20000, about 40000, about 60000, about 80000, about 100000, or about500000 per 1 mm². The portion of the reagents retained each time can bedifferent and controlled to be at a desirable portion depending on thereactions to be performed.

The invention, in various embodiments, contemplates a system forconducting a set of parallel reactions comprising a first surface with aplurality of resolved loci and a capping element with a plurality ofresolved reactor caps. The plurality of resolved loci and the cappingelement with a plurality of resolved reactor caps can be combined toform a plurality of resolved reactors, as described in further detailelsewhere herein. In some cases, the resolved loci of the first surfaceof the first substrate can comprise a coating of reagents. The resolvedloci of the second surface of the second substrate can comprise acoating of reagents. In some embodiments, the coating of reagents can becovalently linked to the first or second surface. In the cases whenthere is a third, a four, a fifth, a sixth, a seventh, a eighth, a ninthor a tenth surface, each surface may comprise a coating of reagents.

The coating of reagents on the first surface or the second surface maycomprise oligonucleotides. The oligonucleotides can be any length asfurther described elsewhere herein, for example at least 25, 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300 bp, or longer. Upon sealingthe resolved loci with the resolved reactor caps, the oligonucleotidesthat are comprised within the coating of reagents may be released. Avariety of reactions can be conducted, for example, the oligonucleotideamplification reaction, PCA, generation of sequencing libraries, orerror correction, inside of the plurality of resolved reactors.

The oligonucleotides can be released from the coated surface by avariety of suitable methods as described in further details elsewhereherein and known in the art, for example by enzymatic cleavage, as iswell known in that art. Examples of such enzymatic cleavage include, butare not limited to, the use of restriction enzymes such as MIyI, orother enzymes or combinations of enzymes capable of cleaving single ordouble-stranded DNA such as, but not limited to, Uracil DNA glycosylase(UDG) and DNA Endonuclease IV. Other methods of cleavage known in theart may also be advantageously employed in the present invention,including, but not limited to, chemical (base labile) cleavage of DNAmolecules or optical (photolabile) cleavage from the surface. PCR orother amplification reactions can also be employed to generate buildingmaterial for gene synthesis by copying the oligonucleotides while theyare still anchored to the substrate. Methods of releasingoligonucleotides are described in P.C.T. Patent Publication No.WO2007137242, and U.S. Pat. No. 5,750,672 which is herein incorporatedby reference in its entirety.

In some cases, the releasing in the releasing the capping element fromthe first surface, and the releasing the capping element from the secondsurface can be performed at a different velocity. The amount of theportion of reagents that is retained upon releasing the capping elementfrom the corresponding surface can be controlled by the velocity or thesurface energy of the capping element and the corresponding surface. Insome cases, the first or second surface comprises a different surfacetension, surface energy, or hydrophobicity with a given liquid, such aswater. In some cases, the resolved loci of the first surface cancomprise a high surface energy, surface tension or hydrophobicity. Thedifference in the surface energy, or hydrophobicity, of the cappingelement and the corresponding surface can be a parameter to control theportion of the reagents that is retained upon release. The volume of thefirst and the second reactions can be different.

In some cases, the air pressure outside of the resolved reactors may begreater than the pressure inside the resolved reactors. In other cases,the air pressure outside of the resolved reactors may be less than thepressure inside of the resolved reactors. The difference in the airpressure outside of the resolved reactors and the inside of the resolvedreactors (or the differential pressure) can affect the sealing of theresolved reactors. By modifying the surface energy or hydrophobicity ofthe first surface and the second surface, the differential pressure mayresult in a curve or straight air/liquid interface within a gap betweenthe first surface and the reactor cap of the second surface.Furthermore, the force needed to release the capping element from thesurface can be controlled by the differential pressure, and thedifferential surface energy. In some cases, the surface can be modifiedto have a differential surface energy and differential pressure suchthat the capping element is capable of being released from the surfaceeasily.

The first or second reaction, or any reaction after the second reactionmay comprise various molecular or biochemical assays as described hereinor any suitable reaction known in the art. In some cases, the first orsecond reaction can comprise polymerase cycling assembly. In some cases,the first or second reaction can comprise enzymatic gene synthesis,annealing and ligation reaction, simultaneous synthesis of two genes viaa hybrid gene, shotgun ligation and co-ligation, insertion genesynthesis, gene synthesis via one strand of DNA, template-directedligation, ligase chain reaction, microarray-mediated gene synthesis,solid-phase assembly, Sloning building block technology, or RNA ligationmediated gene synthesis. The reactions or the method for conducting aset of parallel reactions may further comprise cooling the cappingelement, or cooling the first surface (second surface).

The general process work flow of the methods and compositions of thepresent invention using the systems described herein is illustrated inFIG. 8.

Auxiliary Instrumentation

In one aspect, the current invention concerns systems and methods foroligonucleotide synthesis. The system for oligonucleotide synthesis maycomprise a scanning deposition system. The systems for oligonucleotidesynthesis can comprise a first substrate (e.g. oligonucleotide synthesiswafer) having a functionalized surface and a plurality of resolved lociand a inkjet printer, typically comprising a plurality of printheads.Each printhead is typically configured to deposit one of a variety ofbuilding blocks for reactions that are performed in the resolved loci ofa first substrate, e.g. nucleotide building blocks for phosphoramiditesynthesis. The resolved loci of the oligonucleotide synthesis wafer mayreside in microchannels as described in further detail elsewhere herein.The substrate may be sealed within a flow cell, e.g. by providingcontinuous flow of liquids such as those containing necessary reagentsfor the reactions within the resolved loci (e.g. oxidizer in toluene) orsolvents (e.g. acetonitrile) allowing precise control of dosage andconcentration of reagents at the sites of synthesis, e.g. the resolvedloci of an oligonucleotide synthesis wafer. Flow of an inert gas, suchas nitrogen, may be used to dry the substrate, typically throughenhanced evaporation of a volatile substrate. A variety of means, forexample a vacuum source/a depressurizing pump or a vacuum tank, can beused to create reduced relative pressure (negative pressure) or vacuumto improve drying and reduce residual moisture amounts and any liquiddroplets on the surface. Accordingly, the pressure immediatelysurrounding the substrates or the resolved loci thereof may measure tobe about or less than about 100, 75, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2,1, 0.5, 0.1, 0.05, 0.01 mTorr, or less.

FIG. 3 illustrates an example of a system for oligonucleotide synthesis.Accordingly, an oligonucleotide synthesis wafer is configured to providethe resolved loci for oligonucleotide synthesis with necessary bulkreagents through an inlet manifold and, optionally an outlet manifold.for the bulk reagents can include any suitable reagents, carriers,solvents, buffers, or gasses for oligonucleotide synthesis that iscommonly needed among a plurality of resolved loci in variousembodiments, such as oxidizer, de-block, acetonitrile or nitrogen gas.The inkjet printer printheads can move in X-Y direction to theaddressable locations of the first substrate. A second substrate, suchas a capping element, as described in further detail elsewhere herein,can move in the Z direction, and if needed, in the X and Y directions,to seal with the first substrate, forming a plurality of resolvedreactors. Alternatively, the second substrate may be stationary. In suchcases, the synthesis substrate may move in the Z direction, and ifnecessary in X and Y directions, to align and seal with the secondsubstrate. The synthesized oligonucleotides can be delivered from thefirst substrate to the second substrate. Suitable amounts of fluids maybe passed through an inlet manifold and the resolved loci of a firstsubstrate, into a second substrate to facilitate the delivery ofreagents from the first substrate/the resolved loci thereof into thesecond substrate. In another aspect, current invention relates to asystem for oligonucleotide assembly comprising wafer handling.

In various embodiments, the present invention makes use of systems forscanning deposition. The scanning deposition systems can comprise aninkjet that can be used to deposit reagents to the resolved loci ormicrowells etched into a substrate. In some embodiments, the scanningdeposition system can use organic solvents or inks. In some cases, thescanning deposition system can comprise a plurality of wafers, such assilicon wafers, typically about 200 mm in diameter. In some cases, theentire system can be place and function in an atmospherically controlledenclosure. The scanning deposition system can comprise a work envelope,a printhead assembly, a flowcell assembly, and/or a service envelope. Insome cases, the printhead assembly can move while the flowcell assemblyremains stationary. The scanning deposition system can comprise one ormore flowcells, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,or more flowcells servicing one or more substrates/wafers, such as 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more substrates/wafers.Wafers can stay fixed within the flowcells. In some cases, the systemcan facilitate alignment of substrates through theta_z automation. Thework envelope can include area comprising scanning direction travel,e.g. about (n−1) Printhead Pitch+Wafer Diameter=9*20 mm+200 mm=380 mm,in one particular embodiment. Suitable working envelopes can beenvisioned with equivalent setups. The service envelope may compriseprintheads that are parked for servicing. In some cases, the serviceenvelope can be environmentally isolated from a larger box. In variousembodiments, the systems for the methods and compositions describedherein comprise scanning deposition systems for oligonucleotidesynthesis, oligonucleotide assembly, or more generally for themanufacturing of reagents.

The plurality of resolved loci and the plurality of resolved reactorcaps may be located on microstructures that have interconnectivity orfluidic communications. Such fluidic communications allow washing andperfusing new reagents as droplets or using continuous flow, fordifferent steps of reactions. The fluid communication microchannels maycontain inlets and outlets to and/or from the plurality of resolved lociand the plurality of resolved reactors. The inlets and/or outlets can bemade with any known methods in the art. For example, the inlets and/oroutlets can be provided on a front side and the back side of thesubstrate. Methods of creating the inlets and/or outlets are describedin U.S. Patent Publication No. US 20080308884 A1, which is hereinincorporated by reference in its entirety, an may comprise makingsuitable microstructural components by lithographic and etchingprocesses on a front side; drilling holes from the back side of saidsubstrate in precise alignment with the microstructures on the frontside, to provide inlets and/or outlets to and/or from saidmicromechanical structure. The inlets and/or outlets may be Hele-Shawtype flowcells, with fluid flowing in a thin gap fed by a manifold. Asillustrated in FIG. 9 part A, the substrates described herein, may formpart of a flowcell. The flowcell can be closed by sliding a lid over thetop of the substrate (i.e. wafer) and can be clamped into place forminga pressure tight seal around the edge of the substrate. In someembodiments, the seal may be adequate to seal against vacuum or about 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 atmospheres of pressure. Reagents can beintroduced into a thin gap underneath the substrate (i.e. wafer) andflow up through the substrate. Reagents can then be collected in thetapered waste collector as illustrated in FIG. 9 part B. After a finalsolvent wash step, in some embodiments, the wafer can be drained out,e.g. through the bottom of the assembly and then purged with nitrogen.The chamber can be then pulled down to a vacuum to dry out the remainingsolvent in any microstructures reducing the residual liquids or moistureto less than 50%, 30%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or less by volume. Thechamber can be then pulled down to a vacuum to reduce the pressuresurrounding the substrate to be less than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 25, 50, 75, 100, 200, 300, 400, 500 or 1000 mTorr. In somecases, the chamber can be filled with nitrogen subsequent to the vacuumstep and the roof can be slid open again to allow access by auxiliaryparts of the system, for example a printer. In some cases, the flowcellcan be opened. The substrate/wafer can be mounted with the wastemanifold displaced sideways, as illustrated in FIG. 9 part B. Thisset-up can allow easier inkjet access to the wafer. At this point thereagents can be deposited into the microwells. In some embodiments, thelids of the resolved enclosures (i.e. flowcells) can serve as a wastecollector, and the liquid of reagents may flow thereto. The arrows inFIG. 9 parts B and C represent an exemplary flow direction for thereagents. In some cases, reagents can enter through the thin gap on thebottom, passing through the holes in the substrate (e.g. a siliconwafer), and being collected in the waste collector as illustrated inFIG. 9 part C. In some cases, gas may be purged through an upper orbottom manifold to drive liquid out, e.g. through the bottom or top ofthe flowcell. An exit or inlet port can be connected to vacuum tocomplete drying. The vacuum port can be connected to the waste side orthe inlet side, as illustrated in FIG. 10 parts A-C. In someembodiments, there can be a plurality of pressure release holes thatpass through the substrate (i.e. wafer). The plurality of holes can bemore than a about 1000, 5000, 10,000, 50,000, 100,000, 500,000,1,000,000 or 2,000,000. In some cases, the plurality of holes can bemore than 5 millions. In some cases, the microstructures for synthesisas described in further detail elsewhere herein serve as pressurerelease holes. These holes can allow gas to pass through from one sideof the wafer as the resolved enclosures are evacuated to dry down thesubstrate. In some cases, for example if the air is driven out of thewaste collector side, the air pressure of the waste collector side,P_(waste), may be maintained at substantially the same level as the airpressure of the inlet side, P_(inlet). In some embodiments, a port thatconnects the inlet manifold to the waste collector can be used. Thus, aplurality of the steps described herein, such as scanning, depositing,flooding, washing, purging, and/or drying, can be performed withouttransporting the wafer substrates.

The resolved reactors formed by sealing the first substrate and thesecond substrate may be enclosed in chambers with controlled humidity,air content, vapor pressure, and/or pressure forming an assembly with acontrolled environment. In some embodiments, the humidity of thechambers can be saturated or about 100% to prevent liquid evaporationfrom the resolved reactors during the reactions. For example, thehumidity can be controlled to about, less than about, or more than about100%, 99.5%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94%,93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%,75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% or 25%.

Systems described herein, such as those with controlled environmentassemblies described above may include a vacuum device/chuck and/or atemperature control system operatively connected with the plurality ofresolved reactors. The substrates may be positioned on a vacuum chuck.The vacuum chuck may include surface irregularities positioned directlyunderneath the substrate. In various embodiments, the surfaceirregularities may comprise channels or recesses. The vacuum chuck maybe in fluid communication with the substrate for drawing gas out of thespaces defined by the channels. Methods of maintaining the substrate onvacuum device are described in further detail in U.S. Pat. No.8,247,221, which is herein incorporated by reference in its entirety.

In various embodiments, the substrate (e.g. a silicon wafer) may bepositioned onto a chuck, such as the vacuum chuck described above. FIG.10 parts A-C exemplifies a system assembly of a single groove vacuumchuck and a sintered metal piece in between the substrate and thetemperature control device. The vacuum chuck can comprise a singlegroove with suitable dimensions to hold a substrate. In someembodiments, the vacuum chuck is designed such that a substrate can beheld in place during one or more of the methods described herein. Thevacuum chuck, illustrated in FIG. 10 part A as an example, comprises asingle 1-5 mm groove with approximately 198 mm in diameter. In somecases, single groove vacuum chuck designs can be used to provideimproved heat transfer to the substrate. FIG. 10 part B illustrates asintered metal insert that is situated in between the substrate (e.g.silicon wafer) and the vacuum chuck, being fixed in place withadhesives. In some embodiments, the chuck can be an electrostatic chuck,as further described in U.S. Pat. No. 5,530,516, which is hereinincorporated by reference in its entirety.

The plurality of resolved reactor caps can be aligned with the pluralityof resolved loci on the first surface forming a temporary seal betweenthe first surface and the capping element using any methods that areknown in the art, as described in the U.S. Pat. No. 8,367,016 andEuropean Patent No. EP 0126621 B1, both of which are herein incorporatedby reference in their entirety. For example, for a substrate with aplurality of resolved loci having x, y, and z dimensions and a locusdepth center point located along the z dimension, the locus depth centerpoint can be located a known z dimension distance from a fiducialmarking embedded within the substrate. The substrate can be placedwithin an imaging system that can include an optical device capable ofdetecting the fiducial marking. The optical device can define an opticalpath axially aligned with the z dimension and can have a focal planeperpendicular to the optical path. When the focal plane is moved alongthe optical path, the fiducial marking can be maximally detected whenthe focal plane is at the z depth in comparison to when the focal planeis not substantially in-plane with the z depth. Fiducial markings can beselectively placed in a suitable spatial arrangement on a firstsubstrate, for example a synthesis wafer comprising a plurality ofresolved loci, and/or the second substrate, for example a reactorelement comprising a plurality of capping elements. In some embodiments,the global alignment fiducial marking can be formed close to a resolvedlocus. Depending upon the application, there may be variations,alternatives, and modifications. For example, two of the fiducialmarkings may be within a vicinity of the resolve loci and the thirdfiducial marking may be at the edge of the substrate. For anotherexample, the pattern of the microstructures in substrates describedherein may itself be selected in a recognizable fashion suitable foralignment, for example in an asymmetric pattern, and can be used foralignment. In some cases, the fiducial marking serves as an alignmentpoint to correct for depth of field or other optical characteristics.U.S. Pat. No. 4,123,661, which is herein incorporated by reference inits entirety discloses electronic beam alignment make on a substrate,the marks being adjacent each other but separated by a distance so thatthe rising and falling slopes of the marks can be detected by a videosignal, hence allowing alignments.

The system may comprise a heating component, a cooling component, or atemperature controlled element (e.g., a thermal cycling device). Invarious embodiments, a thermal cycling device for use with a pluralityof resolved reactors may be configured to perform nucleic acidamplification or assembly, such as PCR or PCA or any other suitablenucleic acid reaction described herein or known in the art. Thetemperature can be controlled such that the temperatures within thereactors can be uniform and heat can be conducted quickly. In variousembodiments, the systems described herein may have detection componentsfor end-point or real-time detection from the reactors or the individualmicrostructures within substrates, for example during oligonucleotidesynthesis, gene assembly or nucleic acid amplification.

Any of the systems described herein, may be operably linked to acomputer and may be automated through a computer either locally orremotely. Computers and computer systems for the control of the systemcomponents described herein are further described elsewhere herein.

Primary Compositions—Oligonucleotides

As used herein, the terms “preselected sequence”, “predefined sequence”or “predetermined sequence” are used interchangeably. The terms meanthat the sequence of the polymer is known and chosen before synthesis orassembly of the polymer. In particular, various aspects of the inventionare described herein primarily with regard to the preparation of nucleicacids molecules, the sequence of the oligonucleotide or polynucleotidebeing known and chosen before the synthesis or assembly of the nucleicacid molecules. In one embodiment, oligonucleotides are short nucleicacid molecules. For example, oligonucleotides may be from about 10 toabout 300 nucleotides, from about 20 to about 400 nucleotides, fromabout 30 to about 500 nucleotides, from about 40 to about 600nucleotides, or more than about 600 nucleotides long. Those of skill inthe art appreciate that the oligonucleotide lengths may fall within anyrange bounded by any of these values (e.g., from about 10 to about 400nucleotides or from about 300 to about 400 nucleotides etc.). Suitablyshort or long oligonucleotides may be used as necessitated by thespecific application. Individual oligonucleotides may be designed tohave a different length from another in a library. Oligonucleotides canbe relatively short, e.g. shorter than 200, 100, 80, 60, 50, 40, 30, 25,20, 15, 12, 10, 9, 8, 7, 6, 5, or 4 nucleotides, more particularly.Relatively longer oligonucleotides are also contemplated; in someembodiments, oligonucleotides are longer than or equal to 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80,90, 100, 125, 150, 200, 250, 300, 350, 400, 500, 600 nucleotides, orlonger. Typically, oligonucleotides are single-stranded DNA or RNAmolecules.

In one aspect of the invention, a device for synthesizing a plurality ofnucleic acids having a predetermined sequence is provided. The devicecan include a support having a plurality of features, each featurehaving a plurality of oligonucleotides. In some embodiments, theplurality oligonucleotides having a predefined sequence are immobilizedat different discrete features of a solid support. In some embodiments,the oligonucleotides are single-stranded. In some embodiments, theplurality of oligonucleotide sequences may comprise degeneratesequences. In some embodiments, the oligonucleotides are support-bound.In some embodiments, the device comprises a solid support having aplurality of spots or features, and each of the plurality of spotsincludes a plurality of support-bound oligonucleotides. In someembodiments, the oligonucleotides are covalently linked through their 3′end to the solid support. Yet, in other embodiments the oligonucleotidesare covalently linked through their 5′ end to the solid support.

In some embodiments, the surface or support-bound oligonucleotides areimmobilized through their 3′ end. It should be appreciated that by 3′end, it is meant the sequence downstream to the 5′ end, for example 2,3, 4, 5, 6, 7, 10, 15, 20 nucleotides or more downstream from the 5′end, for another example on the 3′ half, third, or quarter of thesequence, for yet another example, less than 2, 3, 4, 5, 6, 7, 10, 15,or 20 nucleotides away from the absolute 3′ end and by 5′ end it ismeant the sequence upstream to the 3′ end, for example 2, 3, 4, 5, 6, 7,10, 15, 20 nucleotides or more upstream from the 3′ end, for anotherexample on the 5′ half, third, or quarter of the sequence, for yetanother example, less than 2, 3, 4, 5, 6, 7, 10, 15, or 20 nucleotidesaway from the absolute 5′ end. For example, an oligonucleotide may beimmobilized on the support via a nucleotide sequence (e.g., a degeneratebinding sequence), a linker or spacer (e.g., a moiety that is notinvolved in hybridization). In some embodiments, the oligonucleotidecomprises a spacer or linker to separate the oligonucleotide sequencefrom the support. Useful spacers or linkers include photocleavablelinkers, or other traditional chemical linkers. In one embodiment,oligonucleotides may be attached to a solid support through a cleavablelinkage moiety. For example, the solid support may be functionalized toprovide cleavable linkers for covalent attachment to theoligonucleotides. The linker moiety may be of six or more atoms inlength. Alternatively, the cleavable moiety may be within anoligonucleotide and may be introduced during in situ synthesis. A broadvariety of cleavable moieties are available in the art of solid phaseand microarray oligonucleotide synthesis (see e.g., Pon, R., MethodsMol. Biol. 20:465-496 (1993); Verma et al, Annu. Rev. Biochem. 67:99-134(1998); U.S. Pat. Nos. 5,739,386, 5,700,642 and 5,830,655; and U.S.Patent Publication Nos. 2003/0186226 and 2004/0106728). A suitablecleavable moiety may be selected to be compatible with the nature of theprotecting group of the nucleoside bases, the choice of solid support,and/or the mode of reagent delivery, among others. In an exemplaryembodiment, the oligonucleotides cleaved from the solid support containa free 3′-OH end. Alternatively, the free 3′-OH end may also be obtainedby chemical or enzymatic treatment, following the cleavage ofoligonucleotides. In various embodiments, the invention relates tomethods and compositions for release of support or surface boundoligonucleotides into solution. The cleavable moiety may be removedunder conditions which do not degrade the oligonucleotides. Preferablythe linker may be cleaved using two approaches, either simultaneouslyunder the same conditions as the deprotection step or subsequentlyutilizing a different condition or reagent for linker cleavage after thecompletion of the deprotection step.

In other embodiments, the oligonucleotides are in solution. For example,oligonucleotides may be provided within a discrete volume such as adroplet or microdroplet at different discrete features. In someembodiments, discrete microvolumes of between about 0.5 pL and about 100nL may be used. However, smaller or larger volumes may be used. In someembodiments, a suitable dispenser or continuous flow, such as flowthrough microstructures that is actuated by a pump, may be used fortransferring volumes of less than 100 nL, less than 10 nL, less than 5nL, less than 100 pL, less than 10 pL, or less than 0.5 pL to andbetween microstructures of substrates described herein. For example,small volumes from one or more microstructures of an oligonucleotidesynthesis wafer may be dispensed into a reactor cap of a capping elementby pushing a fluid through the oligonucleotide synthesis wafer.

In some embodiments, a plurality of nucleotide acid constructs areprovided at different features of the support. In some embodiments, thenucleic acid constructs, including short oligonucleotides andlonger/assembled polynucleotides, are partially double-stranded orduplex oligonucleotides. As used herein, the term “duplex” refers to anucleic acid molecule that is at least partially double-stranded. Theterms “nucleoside” or “nucleotide” are intended to include thosemoieties which contain not only the known purine and pyrimidine bases,but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, acylatedpurines or pyrimidines, alkylated riboses or other heterocycles or anyother suitable modifications described herein or otherwise known in theart. In addition, the terms “nucleoside” and “nucleotide” include thosemoieties that contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, or are functionalized as ethers, amines, or the like.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” refer to nucleosides and nucleotides containing not onlythe conventional purine and pyrimidine bases, i.e., adenine (A), thymine(T), cytosine (C), guanine (G) and uracil (U), but also protected formsthereof, e.g., wherein the base is protected with a protecting groupsuch as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl or benzoyl,and purine and pyrimidine analogs. Suitable analogs will be known tothose skilled in the art and are described in the pertinent texts andliterature. Common analogs include, but are not limited to,1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine,8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylanminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine and 2,6-diaminopurine. In addition, the terms “nucleoside”and “nucleotide” include those moieties that contain not onlyconventional ribose and deoxyribose sugars, but other sugars as well.Modified nucleosides or nucleotides also include modifications on thesugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen atoms or aliphatic groups, or are functionalizedas ethers, amines, or the like.

As used herein, the term “oligonucleotide” shall be generic topolydeoxynucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), to any other type ofpolynucleotide that is an N-glycoside of a purine or pyrimidine base,and to other polymers containing nonnucleotidic backbones (for examplePNAs), providing that the polymers contain nucleobases in aconfiguration that allows for base pairing and base stacking, such as isfound in DNA and RNA. Thus, these terms include known types ofoligonucleotide modifications, for example, substitution of one or moreof the naturally occurring nucleotides with an analog, inter-nucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoramidates, carbamates,etc.), with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalkylphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.). There is no intended distinction in length between theterm “polynucleotide” and “oligonucleotide,” and these terms will beused interchangeably.

The term “attached,” as in, for example, a substrate surface having amoiety “attached” thereto, includes covalent binding, adsorption, andphysical immobilization. The terms “binding” and “bound” are identicalin meaning to the term “attached.”

In various embodiments, the invention relates to the synthesis, such aschemical synthesis, of molecules other than nucleic acids. The terms“peptide,” “peptidyl” and “peptidic” as used throughout thespecification and claims are intended to include any structure comprisedof two or more amino acids. For the most part, the peptides in thepresent arrays comprise about 5 to 10,000 amino acids, preferably about5 to 1000 amino acids. The amino acids forming all or a part of apeptide may be any of the twenty conventional, naturally occurring aminoacids, i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid(E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I),lysine (K), leucine (L), methionine (M), asparagine (N), proline (P),glutamine (Q), arginine (R), serine (S), threonine (T), valine (V),tryptophan (W), and tyrosine (Y). Any of the amino acids in the peptidicmolecules forming the present arrays may be replaced by anon-conventional amino acid. In general, conservative replacements arepreferred. Conservative replacements substitute the original amino acidwith a non-conventional amino acid that resembles the original in one ormore of its characteristic properties (e.g., charge, hydrophobicity,stearic bulk; for example, one may replace Val with Nval). The term“non-conventional amino acid” refers to amino acids other thanconventional amino acids, and include, for example, isomers andmodifications of the conventional amino acids (e.g., D-amino acids),non-protein amino acids, post-translationally modified amino acids,enzymatically modified amino acids, constructs or structures designed tomimic amino acids (e.g., α,α-disubstituted amino acids, N-alkyl aminoacids, lactic acid, β-alanine, naphthylalanine, 3-pyridylalanine,4-hydroxyproline, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, and nor-leucine), and peptideshaving the naturally occurring amide —CONH— linkage replaced at one ormore sites within the peptide backbone with a non-conventional linkagesuch as N-substituted amide, ester, thioamide, retropeptide (—NHCO—),retrothioamide (—NHCS—), sulfonamido (—SO2NH—), and/or peptoid(N-substituted glycine) linkages. Accordingly, the peptidic molecules ofthe array include pseudopeptides and peptidomimetics. The peptides ofthis invention can be (a) naturally occurring, (b) produced by chemicalsynthesis, (c) produced by recombinant DNA technology, (d) produced bybiochemical or enzymatic fragmentation of larger molecules, (e) producedby methods resulting from a combination of methods (a) through (d)listed above, or (f) produced by any other means for producing peptides.

The term “oligomer” is meant to encompass any polynucleotide orpolypeptide or other chemical compound with repeating moieties such asnucleotides, amino acids, carbohydrates and the like.

In some examples, the device has at least 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 15, 18, 20, 25, 30, 40, 50, 100, 1,000, 4,000, 10,000, 100,000,1,000,000, or more different features (or “regions” or “spots”) at aparticular location (i.e., an “address”). It should be appreciated thata device may comprise one or more solid supports. Each addressablelocation of a device may hold a different composition, such as adifferent oligonucleotide. Alternatively, groups of addressable locationof a device may hold wholly or substantially similar compositions, e.g.oligonucleotides, that are different from those held in other groups ofmicrostructures of a device.

The number of each oligonucleotide, which may be prepared by methods ofthe invention in individually addressable locations and/or in mixedpopulations can range from five to 500,000, from 500 to 500,000, from1,000 to 500,000, from 5,000 to 500,000, from 10,000 to 500,000, from20,000 to 500,000, from 30,000 to 500,000, from 5,000 to 250,000, from5,000 to 100,000, from five to 5,000, from five to 50,000, from 5,000 to800,000, from 5,000 to 1,000,000, from 5,000 to 2,000,000, from 10,000to 2,000,000, from 20,000 to 1,000,000, from 30,000 to 2,000,000, etc.In various embodiments, about or more than about 5, 10, 20, 50, 100,500, 1000, 10000, 100000, 1000000, 10000000, 100000000, or more copiesof each oligonucleotide can be synthesized. In some cases, less than100000000, 10000000, 1000000, 100000, 10000, 1000, 100, or fewer copiesof an oligonucleotide may be synthesized.

Oligonucleotide phosphorothioates (OPS) are modified oligonucleotideswhere one of the oxygen atoms in the phosphate moiety is replaced bysulfur. Phosphorothioates having sulfur at a non-bridging position arewidely used. OPS are substantially more stable towards hydrolysis bynucleases. This property renders OPS to be an advantageous candidate tobe used as antisense oligonucleotides in in vitro and in vivoapplications comprising extensive exposure to nucleases. Similarly, toimprove the stability of siRNA, at least one phosphorothioate linkage isoften introduced at the 3′-terminus of sense and/or antisense strands.In some embodiments, methods and compositions of the invention relate tothe de novo/chemical synthesis of OPSs. The synthesis of a large numberof OPSs may be carried out in parallel using the methods andcompositions described herein.

Amplification of Single Stranded Nucleic Acids

In various embodiments, the methods and systems relate to amplificationof single stranded nucleic acids. Accordingly, single stranded nucleicacids, e.g. single stranded DNA (ssDNA), can be amplified in an isolatedsample, in a plurality of samples in parallel or in a multiplexed formathaving a plurality of different single stranded nucleic acids within thesame sample. The plurality of samples that can be amplified in parallelformat may be at least or about at least 1, 2, 3, 4, 5, 10, 20, 25, 50,55, 100, 150, 200, 250, 300, 350, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, or more. The plurality of samples that can beamplified in parallel format may be between 1-1000, 2-950, 3-900, 4-850,5-800, 10-800, 20-750, 25-700, 30-650, 35-600, 40-550, 45-500, 50-450,55-400, 60-350, 65-250, 70-200, 75-150, 80-100. Those of skill in theart will appreciate that the plurality of samples that can be amplifiedin parallel format may fall between any ranges, bound by any of thesevalues, for example 3-800. The number of multiplexed amplificationreactions may be at least or about at least 1, 2, 3, 4, 5, 10, 20, 25,50, 100, or more. The number of multiplexed amplification reactions maybe between 1-100, 2-50, 3-25, 4-20, 5-10. Those of skill in the art willappreciate that the number of multiplexed amplification reactions mayfall within any range bound by any of these values, for example 3-100.

The number of different single stranded nucleic acids within the samesample can be at least or about at least 1, 2, 3, 10, 50, 100, 150, 200,1000, 10000, 100000, or more. The number of different single strandednucleic acid within the same sample can be at most or about at most10000, 10000, 1000, 200, 150, 100, 50, 10.3, 2.1, or less. The number ofdifferent single stranded nucleic acids within the same sample can bebetween 1-100000, 2-10000, 3-1000, 10-200, 50-100. Those of skill in theart appreciate that the number of different single stranded nucleic acidwithin the same sample can be between any of these ranges, bound by anyof these values, for example 3-100.

The single stranded target nucleic acids may be at least or about atleast 10, 20, 50, 100, 200, 500, 1000, 3000, or more nucleotides long.The single stranded target nucleic acids may be at most or about at most3000, 1000, 500, 200, 100, 50, 20, 10, or less, nucleotides long. Thesingle stranded target nucleic acids may be between 50-500, 75-450, or100-400 nucleotides long. Those of skill in the art appreciate thatlength of the single stranded target nucleic acids may fall within anyrange bound by any of these values, for example between 50-1000.

Referring now to FIG. 64, a single stranded target nucleic acid may beflanked with one or more adaptor hybridization sequences. These adaptorhybridization sequences sequences may be at least or about at least 12,13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides long. These adaptorhybridization sequences sequences may be at least or about at least 20,19, 18, 17, 16, 15, 14, 13, 12, or fewer nucleotides long. The adaptorhybridization sequences may be between 15-20, 16-19, 17-18 nucleotideslong. Those of skill in the art appreciate that length the adaptorhybridization sequences may fall between a range bound by any of thesevalues, for example between 15-17, 12-20, or 13, 25. The adaptorhybridization sequences may be shared by a plurality of nucleic acidswithin a sample, wherein such plurality of single stranded nucleic acidshave varying single stranded target nucleic acid regions. Multiplegroups of single stranded nucleic acids, each group having differentadaptor hybridization sequences, may coexist within a sample and besubjected to the amplification methods described herein. The differentadaptor hybridization sequences may differ from each other by at leastor at least about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more,nucleotides. The different adaptor hybridization sequences may differfrom each other by at most or at most about 50, 45, 40, 35, 30, 25, 20,15, 10, 5, 2, 1, or fewer nucleotides. The different adaptorhybridization sequences may differ from each other by a number ofnucleotides between 1-50, 2-45, 5-40, 10-35, 15-25, or 20-30. Those ofskill in the art appreciate that, the different adaptor hybridizationsequences may differ from each other by a number of nucleotides thatfalls in any ranges bound by any of these values, for example between2-50. Thus, a single universal adaptor may be used for a number ofsingle stranded nucleic acids sharing end sequences such that theuniversal adaptor is hybridizable to all of them. A plurality ofadaptors may be used in a sample with a plurality of groups of singlestranded nucleic acids, wherein each of the adaptors is hybridizable tothe end sequences in one or more of the groups. At least or at leastabout 1, 2, 3, 4, 5, 10, 20, 25, 30, 50, 100, or more adaptors may beused in a multiplexed fashion. At most or about at most 100, 50, 30, 25,20, 10, 5, 4, 3, 21.1 or fewer adaptors may be used in a multiplexedfashion. Between 1-100, 2-50, 3-30, 4-25, 5-20, adaptors may be used ina multiplexed fashion. Those of skill in the art appreciate that thenumber of adaptors that may be used in a multiplexed fashion may fallwithin any ranges, bound by any of these values, for example between2-30. A first sequence on an adaptor may hybridize to the 5′ end of asingle stranded nucleic acid and a second sequence on the adaptor mayhybridize to the 3′ end of the same single stranded nucleic acid,facilitating the circularization of the single stranded nucleic acid.

The single stranded nucleic acids may be circularized upon hybridizationwith an adaptor. The circularized single stranded nucleic acid may bejoined at its 5′ and 3′ ends, forming a contiguous circle. Variousligation methods and enzymes are suitable for the reaction as describedelsewhere herein and otherwise known in the art.

The adaptor can be extended using the circularized single strandednucleic acid as a template. Alternatively, one or more different primersmay be used to anneal elsewhere on the circle in addition or instead ofthe adaptor and can be extended with a polymerase enzyme. The extensionreaction, such as rolling circle amplification, multi-primer rollingcircle amplification or any other suitable extension reaction, canfacilitate the creation of one long and linear single stranded ampliconnucleic acids comprising alternating replicas of the single strandedtemplate nucleic acid and the adaptor hybridization sequences. In someembodiments, the combined replicas of the adaptor hybridizationsequences are copies of the adaptor sequence, or differ by less than 8,7, 6, 5, 4, 3, or 2 nucleotides. These sequences will together bereferred to as “adaptor copies” for ease, but it is understood that theymay refer to a number of different types of sequences generated from theextension reaction using the circle as a template.

One or more auxiliary oligonucleotides may be provided to anneal to thesingle stranded amplicon nucleic acids. The auxiliary oligonucleotidesmay be partially or completely complementary to the adaptor copies. Thehybridization of the auxiliary oligonucleotide to the single strandedamplicon nucleic acid can form alternating single and double strandedregions. The single stranded regions may correspond to replicas of thesingle stranded template nucleic acid sequence. The hybridization of theauxiliary oligonucleotide to the single stranded amplicon nucleic acid,e.g. at adaptor copies, can generate recognition sites for a cleavingagent, such as a restriction endonucleases, e.g. a Type IIS restrictionendonucleases. The sequences can be designed in such a way that thecutting site for the cleaving agent falls at or near the juncture of thesingle and double stranded regions. In some cases, upon cleavage withone or more cleaving agents, a plurality of single stranded replicas ofthe single stranded target nucleic acids will be formed, wherein thesingle stranded target nucleic acids do not contain any portions fromthe adaptor copies, or contain less than 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 nucleotides from the adaptor copies.

The auxiliary oligonucleotides may have an affinity tag, such as biotinor a biotin derivative. The affinity tag may be at the 5′ end, 3′ end,or in the middle of the oligonucleotide. Purification of the auxiliaryoligonucleotides from the sample may be facilitated using an affinitybinding partner on a purification medium, such as streptavidin coatedbeads surfaces, or any other suitable affinity purification method.Cleaved adaptor copies or portion thereof may also be purified alongwith the auxiliary oligonucleotides, facilitated by their hybridizationwith the auxiliary oligonucleotides. In multiplexed reactions using aplurality of adaptors, a plurality of auxiliary oligonucleotides may beused, each hybridizing to a different group of single stranded ampliconnucleic acids, for example at the locations of the adaptor copies.Alternative purification methods, such as HPLC or PAGE purification, maybe used with or without affinity tagged oligonucleotides.

Referring now to FIG. 65 parts A-F, single stranded nucleic acids mayalso be amplified in a similar way to the method described for FIG. 64parts A-F, with the exception that the sequences and the cleaving agentis selected such that the cutting site falls within the adaptor copiessuch that single stranded replicas of the single stranded target nucleicacid sequence are formed with flanking regions. Such flanking regionsmay be reverse complements of the flanking regions of the originalsingle stranded target nucleic acid sequence. Alternatively, dependingon the exact location of the cutting site, they may “shift” nucleotidesfrom one flanking region to the other. In such cases, a reversecomplementary oligonucleotide to the adaptor nucleotide can stilleffectively hybridize to the both ends facilitating another round ofcircularization. Thus, the method illustrated in FIG. 65 parts A-F canbe repeated a plurality of times, such as at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more times, alone or as a precursor reaction tothe method illustrated in FIG. 64 parts A-F, to amplify the singlestranded target nucleic acid. The method illustrated in FIG. 64 partsA-F can be used as a last round to get rid of the flanking regions,leaving behind amplified single stranded copies or replicas of thesingle stranded target nucleic acids.

The extension reaction product, such as a rolling cycle amplificationproduct, comprising single-stranded repeating units of amplified desiredoligonucleotides and adaptor oligonucleotides, may be cleaved within ornear the adaptor oligonucleotides to generate released desiredoligonucleotides, wherein the released desired oligonucleotides may ormay not comprise adaptor nucleotides at the 5′ or 3′ ends of the desiredoligonucleotide. In some embodiments, the cleaving is accomplished atthe very juncture of the single-stranded repeating units of amplifieddesired oligonucleotides and adaptor sequences. In some embodiments, oneor more regions of an adaptor sequence comprise a molecular barcode,protein binding site, restriction endonuclease site, or any combinationthereof. In some embodiments, the amplification product is cleaved withone or more restriction endonucleases at or near a restrictionendonuclease recognition site, wherein the recognition site is locatedwithin an adaptor oligonucleotide sequence. Prior to cleavage with anendonuclease, the amplification product can be hybridized with anauxiliary oligonucleotide comprising a sequence complementary to theadaptor oligonucleotide sequence comprising the restriction endonucleaserecognition site.

The amplification product may be cleaved at the 5′ end of a recognitionsite by Type II endonucleases. The cutting site may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25nucleotides or more upstream from the first nucleotide of therecognition site. The 5′ or 3′ end of a recognition site may form a 0,1, 2, 3, 4, or 5 nucleotide overhang. Blunt Type II endonucleases whichcleave with a 0 nucleotide overhang include MlyI and SchI. ExemplaryType IIS endonucleases which generate 5′ overhangs (e.g., 1, 2, 3, 4, 5nucleotides overhangs) include, but are not limited to, AlwI, BccI,BceAI, BsmAI, BsmFI, FokI, HgaI, PleI, SfaNI, BfuAI, BsaI, BspMI, BtgZI,EarI, BspQI, SapI, SgeI, BceFI, BslFI, BsoMAI, Bst71I, FaqI, AceIII,BbvII, BveI, and LguI. Nicking endonucleases which remove therecognition site and cleave on the 5′ site of the recognition siteinclude, but are not limited to Nb.BsrDI, Nb.BtsI, AspCNI, BscGI,BspNCI, EcoHI, FinI, TsuI, UbaF11I, UnbI, Vpak11AI, BspGI, DrdII,Pfl1108I, and UbaPI.

The amplification product may be cleaved by non-Type IIS endonucleaseswhich cleave at the 5′ end of the recognition site on both strands togenerate a blunt end. The amplification product may be cleaved bynon-Type IIS endonucleases which cleave at the 5′ end of the recognitionsite on one strand and in the middle of the recognition site on theother strand, generating a 5′ overhang. Examples of endonucleases whichgenerate a 5′ overhang include, but are not limited to, BfuCI, DpnII,FatI, MboI, MluCI, Sau3AI, Tsp509I, BssKI, PspGI, StyD4I, Tsp45I, AoxI,BscFI, Bsp143I, BssMI, BseENII, BstMBI, Kzo9I, NedII, Sse9I, TasI,TspEI, AjnI, BstSCI, EcoRII, MaeIII, NmuCI, and Psp6I.

The amplification product may be cleaved by nicking endonucleases whichcleave at the 5′ end of a recognition site to produce a nick. Thenicking site may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides or more upstream fromthe first nucleotide of the recognition site. Exemplary nickingendonucleases include, but are not limited to, Nb.BsrDI, Nb.BtsI,AspCNI, BscGI, BspNCI, EcoHI, FinI, TsuI, UbaF11I, UnbI, Vpak11AI,BspGI, DrdII, Pfl1108I, and UbaPI.

The amplification product may be cleaved at the 3′ end of a recognitionsite by Type IIS endonucleases. The 5′ or 3′ end of a recognition sitemay form a 0, 1, 2, 3, 4, or 5 nucleotide overhang. The cutting site maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25 nucleotides or more downstream from the lastnucleotide of the recognition site. Type IIS endonucleases which cleaveat 0 nucleotides downstream of the last nucleotide of the recognitionsite include MlyI and SchI. Exemplary Type IIS endonucleases whichgenerate 3′ overhangs (e.g., 1, 2, 3, 4, 5 nucleotide overhangs)include, but are not limited to, MnlI, BspCNI, BsrI, BtsCI, HphI, HpyAV,MboII, AcuI, BciVI, BmrI, BpmI, BpuEI, BseRI, BsgI, BsmI, BsrDI, BtsI,EciI, MmeI, NmeAIII, Hin4II, TscAI, Bce83I, BmuI, BsbI, and BscCI.Non-Type II endonucleases which remove the recognition site on onestrand and generate a 3′ overhang or blunt end on the other strandinclude, but are not limited to NlaIII, Hpy99I, TspRI, FaeI, Hin1II,Hsp92II, SetI, TaiI, TscI, TscAI, and TseFI. Nicking endonucleases whichremove the recognition site and cut on the 3′ end of the recognitionsite include Nt.AlwI, Nt.BsmAI, Nt.BstNBI, and Nt.BspQI.

The distance between the recognition site and the cleavage site maydepend on the restriction endonuclease used for cleavage. For example,restriction endonucleases with cutting sites located 1 base pairdownstream or upstream from a recognition site which may efficientlycleave under optimal reaction conditions include, but are not limitedto, AgeI, ApaI, AscI, BmtI, BsaI, BsmBI, BsrGI, DdeI, DraIII, HpaI,MseI, PacI, PciI, PmeI, PvuI, SacII, SapI, Sau3AI, ScaI, SfiI, SmaI,SphI, StuI, and XmaI. Restriction endonucleases with cutting siteslocated 2 base pairs downstream or upstream from a recognition sitewhich may efficiently cleave under optimal reaction conditions include,but are not limited to, AgeI, AluI, ApaI, AscI, BglII, BmtI, BsaI,BsiWI, BsmBI, BsrGI, BssHII, DdeI, DralII, EagI, HpaI, KpnI, MseI,NlaIII, PacI, PciI, PmeI, PstI, PvuI, RsaI, SacII, SapI, Sau3AI, SbfI,ScaI, Sfil, SmaI, SphI, SspI, StuI, StyI, and XmaI. Restrictionendonucleases with cutting sites located 3 base pairs downstream orupstream from a recognition site which may efficiently cleave underoptimal reaction conditions include, but are not limited to, AgeI, AluI,ApaI, AscI, AvrII, BamHI, BglII, BmtI, BsaI, BsiWI, BsmBI, BsrGI,BssHII, DdeI, DralII, EagI, FseI, HindIII, HpaI, KpnI, MfeI, MluI, MseI,NcoI, NdeI, NheI, NlaIII, NsiI, PacI, PciI, PmeI, PstI, RsaI, SacI,SacII, SaII, SapI, Sau3AI, Sbfl, ScaI, Sfil, SmaI, SphI, SspI, StuI,StyI, and XmaI. Restriction endonucleases with cutting sites located 4base pairs downstream or upstream from a recognition site which mayefficiently cleave under optimal reaction conditions include, but arenot limited to, AgeI, AluI, ApaI, AscI, AvrII, BamHI, BglII, BmtI, BsaI,BsiWI, BsmBI, BsrGI, BssHII, ClaI, DdeI, DralII, EagI, EcoRI, FseI,HindIII, HpaI, KpnI, MfeI, MluI, MseI, NcoI, NdeI, NheI, NlaIII, NsiI,PacI, PciI, PmeI, PstI, PvuI, PvuII, RsaI, SacI, SacII, SaII, SapI,Sau3AI, Sbfl, ScaI, Sfil, SmaI, SphI, SspI, StuI, StyI, XhoI, and XmaI.Restriction endonucleases with cutting sites located 5 base pairsdownstream or upstream from a recognition site which may efficientlycleave under optimal reaction conditions include, but are not limitedto, AgeI, AluI, ApaI, AscI, AvrII, BamHI, BglII, BmtI, BsaI, BsiWI,BsmBI, BsrGI, BssHII, ClaI, DdeI, DralII, EagI, EcoRI, EcoRV, FseI,HindIII, HpaI, KpnI, MfeI, MluI, MseI, NcoI, NdeI, NheI, NlaIII, NsiI,NspI, Pad, PciI, PmeI, PstI, PvuI, PvuII, RsaI, SacI, SacII, Sall, SapI,Sau3AI, Sbfl, ScaI, Sfil, SmaI, SphI, SspI, StuI, StyI, XhoI, and XmaI.

The adaptor sequence may comprise one or more restriction recognitionsites. In some embodiments, the recognition site is at least 4, 5, or 6base pairs long. In some embodiments, the recognition site isnon-palindromic. In some embodiments, the adaptor oligonucleotidecomprises two or more recognition sites. Two or more recognition sitesmay be cleaved with one or more restriction enzymes. It will be known toone of skill in the art that the cleavage of two or more recognitionsites with two or more restriction enzymes may be achieved and/orperfected by buffer and reaction temperature optimization. Exemplarypairs of recognition sites in an adaptor sequence include, but are notlimited to, MlyI-MlyI, MlyI-Nt.AlwI, BsaI-MlyI, MlyI-BciVI, andBfuCI-MlyI.

Genes

The methods and compositions of the invention in various embodimentsallow for the construction of gene libraries comprising a collection ofindividually accessible polynucleotides of interest. The polynucleotidescan be linear, can be maintained in vectors (e.g., plasmid or phage),cells (e.g., bacterial cells), as purified DNA, or in other suitableforms known in the art. Library members (variously referred to asclones, constructs, polynucleotides, etc.) can be stored in a variety ofways for retrieval and use, including for example, in multiwell cultureor microtiter plates, in vials, in a suitable cellular environment(e.g., E. coli cells), as purified DNA compositions on suitable storagemedia (e.g., the Storage IsoCodeD IDTM DNA library card; Schleicher &Schuell BioScience), or a variety of other suitable library forms knownin the art. A gene library may comprise at least about 10, 100, 200,300, 400, 500, 600, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7500,10000, 15000, 20000, 30000, 40000, 50000, 60000, 75000, 100000 members,or more. Nucleic acid molecules described herein may be produced inmicroscale quantities (e.g., femtomoles to nanomoles quantities, such asfrom about 0.001 femtomole to about 1.0 nanomole, from about 0.01femtomole to about 1.0 nanomole, from about 0.1 femtomole to about 1.0nanomole, from about 0.001 femtomole to about 0.1 nanomole, from about0.001 femtomole to about 0.01 nanomole, from about 0.001 femtomole toabout 0.001 nanomole, from about 1.0 femtomole to about 1.0 nanomole,from about 1.0 femtomole to about 0.1 nanomole, from about 1.0 femtomoleto about 0.01 nanomole, from about 1.0 femtomole to about 0.001nanomole, from about 10 femtomoles to about 1.0 nanomole, from about 10femtomoles to about 0.001 nanomole, from about 20 femtomoles to about1.0 nanomole, from about 100 femtomoles to about 1.0 nanomole, fromabout 500 femtomoles to about 1.0 nanomole, from about 1 nanomole toabout 800 nanomoles, from about 40 nanomoles to about 800 nanomoles,from about 100 nanomoles to about 800 nanomoles, from about 200nanomoles to about 800 nanomoles, from about 500 nanomoles to about 800nanomoles, from about 100 nanomoles to about 1,000 nanomoles, etc.).Those of skill in the art appreciate that the nucleic acid quantity mayfall within any range bounded by any of these values (e.g., from about0.001 femtomole to about 1000 nanomoles or from about 0.001 femtomole toabout 0.01 femtomole). In general, nucleic acid molecules may beproduced at quantities of about or more than about 0.001, 0.01, 0.1, 1,10, 100, femtomoles, 1, 10, 100 picomoles, 1, 10, 100 nanomoles, 1micromole, or more. In some embodiments, nucleic acid molecules may beproduced at quantities of less than about 1 micromole, 100, 10, 1nanomoles, 100, 10, 1 picomoles, 100, 10, 1, 0.1, 0.001, 0.001femtomoles or less. In some embodiments, nucleic acid molecules may beproduced at concentrations of about or more than about 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, 1000 nM. In someembodiments, the gene library is synthesized/assembled and/or held in aspace that is less than 1000, 100, 10, 1 m³, 100, 10, 1 dm³, 100, 10, 1cm³, or less.

The location of individually accessible members can be available oreasily determined. Individually accessible members may be easilyretrieved from the library.

In various embodiments, the methods and compositions of the inventionallow for production of synthetic (i.e. de novo synthesized) genes.Libraries comprising synthetic genes may be constructed by a variety ofmethods described in further detail elsewhere herein, such as PCA,non-PCA gene assembly methods or hierarchical gene assembly, combining(“stitching”) two or more double-stranded polynucleotides (referred tohere as “synthons”) to produce larger DNA units (i.e., multisynthons orchassis). Libraries of large constructs may involve polynucleotides thatare at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60,70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 kb long orlonger. The large constructs can be bounded by an independently selectedupper limit of about 5000, 10000, 20000 or 50000 base pairs. Thesynthesis of any number of polypeptide-segment encoding nucleotidesequences, including sequences encoding non-ribosomal peptides (NRPs),sequences encoding non-ribosomal peptide-synthetase (NRPS) modules andsynthetic variants, polypeptide segments of other modular proteins, suchas antibodies, polypeptide segments from other protein families,including non-coding DNA or RNA, such as regulatory sequences e.g.promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA,small nucleolar RNA derived from microRNA, or any functional orstructural DNA or RNA unit of interest. The term “gene” as used hereinrefers broadly to any type of coding or non-coding, long polynucleotideor polynucleotide analog.

In various embodiments, the methods and compositions of the inventionrelate to a library of genes. The gene library may comprise a pluralityof subsegments. In one or more subsegments, the genes of the library maybe covalently linked together. In one or more subsegments, the genes ofthe library may encode for components of a first metabolic pathway withone or more metabolic end products. In one or more subsegments, genes ofthe library may be selected based on the manufacturing process of one ormore targeted metabolic end products. The one or more metabolic endproducts comprise a biofuel. In one or more subsegments, the genes ofthe library may encode for components of a second metabolic pathway withone or more metabolic end products. The one or more end products of thefirst and second metabolic pathways may comprise one or more shared endproducts. In some cases, the first metabolic pathway comprises an endproduct that is manipulated in the second metabolic pathway.

In some embodiments, a subsegment of the library may comprise, consistsof, or consists essentially of genes encoding for a part or all of thegenome of a synthetic organism, e.g. a virus or a bacterium. Thus, theterms “gene”, “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably and referto a nucleotide polymer. Unless otherwise limited, the same includeknown analogs of natural nucleotides that can function in a similarmanner (e.g., hybridize) to naturally occurring nucleotides. They can beof polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, intergenic DNA, loci (locus) defined from linkageanalysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA(miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), whichis a DNA representation of mRNA, usually obtained by reversetranscription of messenger RNA (mRNA) or by amplification; DNA moleculesproduced synthetically or by amplification, genomic DNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted by nonnucleotide components. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component.Polynucleotide sequences, when provided, are listed in the 5′ to 3′direction, unless stated otherwise.

The term nucleic acid encompasses double- or triple-stranded nucleicacids, as well as single-stranded molecules. In double- ortriple-stranded nucleic acids, the nucleic acid strands need not becoextensive (i.e., a double-stranded nucleic acid need not bedouble-stranded along the entire length of both strands).

The term nucleic acid also encompasses any chemical modificationthereof, such as by methylation and/or by capping. Nucleic acidmodifications can include addition of chemical groups that incorporateadditional charge, polarizability, hydrogen bonding, electrostaticinteraction, and functionality to the individual nucleic acid bases orto the nucleic acid as a whole. Such modifications may include basemodifications such as 2′-position sugar modifications, 5-positionpyrimidine modifications, 8-position purine modifications, modificationsat cytosine exocyclic amines, substitutions of 5-bromo-uracil, backbonemodifications, unusual base pairing combinations such as the isobasesisocytidine and isoguanidine, and the like.

More particularly, in certain embodiments, nucleic acids, can includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other type of nucleicacid that is an N- or C-glycoside of a purine or pyrimidine base, aswell as other polymers containing nonnucleotidic backbones, for example,polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino(commercially available from the Anti-Virals, Inc., Corvallis, Oreg., asNeugene) polymers, and other synthetic sequence-specific nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA. The term nucleic acid also encompasses linkednucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499,6,670,461, 6,262,490, and 6,770,748, which are incorporated herein byreference in their entirety for their disclosure of LNAs.

As used herein, the term “complementary” refers to the capacity forprecise pairing between two nucleotides. If a nucleotide at a givenposition of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid, then the two nucleic acids areconsidered to be complementary to one another at that position.Complementarity between two single-stranded nucleic acid molecules maybe “partial”, in which only some of the nucleotides bind, or it may becomplete when total complementarity exists between the single-strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

“Hybridization” and “annealing” refer to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of a PCR or otheramplification reactions, or the enzymatic cleavage of a polynucleotideby a ribozyme. A first sequence that can be stabilized via hydrogenbonding with the bases of the nucleotide residues of a second sequenceis said to be “hybridizable” to said second sequence. In such a case,the second sequence can also be said to be hybridizable to the firstsequence.

The term “hybridized” as applied to a polynucleotide refers to apolynucleotide in a complex that is stabilized via hydrogen bondingbetween the bases of the nucleotide residues. The hydrogen bonding mayoccur by Watson Crick base pairing, Hoogstein binding, or in any othersequence specific manner. The complex may comprise two strands forming aduplex structure, three or more strands forming a multi strandedcomplex, a single self hybridizing strand, or any combination of these.The hybridization reaction may constitute a step in a more extensiveprocess, such as the initiation of a PCR reaction, or the enzymaticcleavage of a polynucleotide by a ribozyme. A sequence hybridized with agiven sequence is referred to as the “complement” of the given sequence.

“Specific hybridization” refers to the binding of a nucleic acid to atarget nucleotide sequence in the absence of substantial binding toother nucleotide sequences present in the hybridization mixture underdefined stringency conditions. Those of skill in the art recognize thatrelaxing the stringency of the hybridization conditions allows sequencemismatches to be tolerated.

In general, a “complement” of a given sequence is a sequence that isfully or substantially complementary to and hybridizable to the givensequence. In general, a first sequence that is hybridizable to a secondsequence or set of second sequences is specifically or selectivelyhybridizable to the second sequence or set of second sequences, suchthat hybridization to the second sequence or set of second sequences ispreferred (e.g. thermodynamically more stable under a given set ofconditions, such as stringent conditions commonly used in the art) tohybridization with non-target sequences during a hybridization reaction.Typically, hybridizable sequences share a degree of sequencecomplementarity over all or a portion of their respective lengths, suchas between 25%-100% complementarity, including at least about 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity.

The term “primer” refers to an oligonucleotide that is capable ofhybridizing (also termed “annealing”) with a nucleic acid and serving asan initiation site for nucleotide (RNA or DNA) polymerization underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as DNA orRNA polymerase or reverse transcriptase) in an appropriate buffer and ata suitable temperature. The appropriate length of a primer depends onthe intended use of the primer, but primers are typically at least 7nucleotides long and, more typically range from 10 to 30 nucleotides, oreven more typically from 15 to 30 nucleotides, in length. Other primerscan be somewhat longer, e.g., 30 to 50 or 40-70 nucleotides long. Thoseof skill in the art appreciate that the primer length may fall withinany range bounded by any of these values (e.g., from 7 to 70 or from 50to 70). Oligonucleotides of various lengths as further described hereincan be used as primers or building blocks for amplification and/or geneassembly reactions. In this context, “primer length” refers to theportion of an oligonucleotide or nucleic acid that hybridizes to acomplementary “target” sequence and primes nucleotide synthesis. Shortprimer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. A primer neednot reflect the exact sequence of the template but must be sufficientlycomplementary to hybridize with a template. The term “primer site” or“primer binding site” refers to the segment of the target nucleic acidto which a primer hybridizes. A construct presenting a primer bindingsite is often referred to as a “priming ready construct” or“amplification ready construct”.

A primer is said to anneal to another nucleic acid if the primer, or aportion thereof, hybridizes to a nucleotide sequence within the nucleicacid. The statement that a primer hybridizes to a particular nucleotidesequence is not intended to imply that the primer hybridizes eithercompletely or exclusively to that nucleotide sequence.

Oligonucleotide Synthesis

Oligonucleotides synthesized on the substrates described herein maycomprise greater than about 100, preferably greater than about 1000,more preferably greater than about 16,000, and most preferably greaterthan 50,000 or 250,000 or even greater than about 1,000.000 differentoligonucleotide probes, preferably in less than 20, 10, 5, 1, 0.1 cm²,or smaller surface area.

A method of quickly synthesizing n-mer, such as about or at least about100-, 150-, 200, 250-, 300, 350-, or longer nucleotide, oligonucleotideson a substrate is further described herein in various embodiments. Themethod can use a substrate with resolved loci that are functionalizedwith a chemical moiety suitable for nucleotide coupling. Standardphosphoramidite chemistry can be used in some cases. Accordingly, atleast two building blocks are coupled to a plurality of growingoligonucleotide chains each residing on one of the resolved loci at afast rate, such as a rate of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotidesper hour, or more. In some embodiments, adenine, guanine, thymine,cytosine, or uridine building blocks, or analogs/modified versionsthereof are used as described in further detail elsewhere herein. Insome cases, the added building blocks comprise dinucleotides,trinucleotides, or longer nucleotide based building blocks, such asbuilding blocks containing about or at least about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or morenucleotides. In some embodiments, large libraries of n-meroligonucleotides are synthesized in parallel on substrate, e.g. asubstrate with about or at least about 100, 1000, 10000, 100000,1000000, 2000000, 3000000, 4000000, 5000000 resolved loci hostingoligonucleotide synthesis. Individual loci may host synthesis ofoligonucleotides that are different from each other. In someembodiments, during the flow of phosphoramidite chemistry, e.g. aprocess with coupling, capping, oxidation, and deblocking steps, reagentdosage can be accurately controlled through cycles ofcontinuous/displacing flow of liquids and vacuum drying steps, such as avacuum drying step prior to coupling of new building blocks. Thesubstrate may comprise vias, such as at least about 100, 1000, 10000,100000, 1000000, or more vias providing fluid communication between afirst surface of the substrate and a second surface of the substrate.Substrates may be kept in place during one or all of the steps within aphosphoramidite chemistry cycle and flow reagents may be passed throughthe substrate.

A common method for the preparation of synthetic nucleic acids is basedon the fundamental work of Caruthers and is known as the phosphoramiditemethod (M. H. Caruthers, Methods in Enzymology 154, 287-313, 1987;incorporated herein by reference in its entirety). The sequence of theresultant molecules can be controlled by the order of synthesis. Othermethods, such as the H-phosphonate method, serve the same purpose ofsuccessive synthesis of a polymer from its subunits.

Typically, the synthesis of DNA oligomers by the methods of theinvention may be achieved through traditional phosphoramidite chemistry.Phosphoramidite based chemical synthesis of nucleic acids is well knownto those of skill in the art, being reviewed in Streyer, Biochemistry(1988) pp 123-124 and U.S. Pat. No. 4,415,732, herein incorporated byreference. Phosporamidite reagents, including B-cyanoethyl (CE)phosphoramidite monomers and CPG (controlled porous glass) reagentsusable with the invention may be purchased from numerous commercialsources, including American International Chemical (Natick Mass.), BDBiosciences (Palo Alto Calif.), and others.

In various embodiments, the chemical synthesis of nucleic acids isoverwhelmingly performed using variations of the phosphoramiditechemistry on solid surfaces (Beaucage S L, Caruthers M H.Deoxynucleoside phosphoramidites—a new class of key intermediates fordeoxypolynucleotide synthesis. Tetrahedron Lett. 1981; 22:1859-1862;Caruthers M H. Gene synthesis machines—DNA chemistry and its uses.Science. 1985; 230:281-285.), both of which are incorporated herein byreference in their entirety.

For instance, phosphoramidite based methods can be used to synthesizeabundant base, backbone and sugar modifications of deoxyribo- andribonucleic acids, as well as nucleic acid analogs (Beaucage S L, Iyer RP. Advances in the synthesis of oligonucleotides by the phosphoramiditeapproach. Tetrahedron. 1992; 48:2223-2311; Beigelman L, Matulic-AdamicJ, Karpeisky A, Haeberli P, Sweedler D. Base-modified phosphoramiditeanalogs of pyrimidine ribonucleosides for RNA structure-activitystudies. Methods Enzymol. 2000; 317:39-65; Chen X, Dudgeon N, Shen L,Wang J H. Chemical modification of gene silencing oligonucleotides fordrug discovery and development. Drug Discov. Today. 2005; 10:587-593;Pankiewicz K W. Fluorinated nucleosides. Carbohydrate Res. 2000;327:87-105; Lesnikowski Z J, Shi J, Schinazi R F. Nucleic acids andnucleosides containing carboranes. J. Organometallic Chem. 1999;581:156-169; Foldesi A, Trifonova A, Kundu M K, Chattopadhyaya J. Thesynthesis of deuterionucleosides. Nucleosides Nucleotides Nucleic Acids.2000; 19:1615-1656; Leumann C J. DNA Analogues: from supramolecularprinciples to biological properties. Bioorg. Med. Chem. 2002;10:841-854; Petersen M, Wengel J. LNA: a versatile tool for therapeuticsand genomics. Trends Biotechnol. 2003; 21:74-81; De Mesmaeker A, AltmannK-H, Waldner A, Wendeborn S. Backbone modifications in oligonucleotidesand peptide nucleic acid systems. Curr. Opin. Struct. Biol. 1995;5:343-355), all of which are incorporated herein by reference in theirentirety.

The phosphoramidite chemistry has been adapted for in situ synthesis ofDNA on solid substrates, e.g. microarrays. Such synthesis is typicallyachieved by spatial control of one step of the synthesis cycle, whichresults in thousands to hundreds of thousands of unique oligonucleotidesdistributed in a small area, e.g. an area of a few square centimeters.The areas and substrates architectures for the synthesis ofoligonucleotides are further described elsewhere herein in greaterdetail. Suitable methods used to achieve spatial control can include (i)control of the coupling step by inkjet printing (Agilent, Protogene;Hughes T R, Mao M, Jones A R, Burchard J, Marton M J, Shannon K W,Lefkowitz S M, Ziman M, Schelter J M, Meyer M R, et al. Expressionprofiling using microarrays fabricated by an ink-jet oligonucleotidesynthesizer. Nat. Biotechnol. 2001; 19:342-347; Butler J H, Cronin M,Anderson K M, Biddison G M, Chatelain F, Cummer M, Davi D J, Fisher L,Frauendorf A W, Frueh F W, et al. In situ synthesis of oligonucleotidearrays by using surface tension. J. Am. Chem. Soc. 2001; 123:8887-8894)or physical masks (Southern E M, Maskos U, Elder J K. Analyzing andcomparing nucleic acid sequences by hybridization to arrays ofoligonucleotides: evaluation using experimental models. Genomics. 1992;13:1008-1017.), (ii) control of the 5′-hydroxyl deblock step byclassical (Affymetrix; Pease A C, Solas D, Sullivan E J, Cronin M T,Holmes C P, Fodor S P A. Light-generated oligonucleotide arrays forrapid dna-sequence analysis. Proc. Natl Acad. Sci. USA. 1994;91:5022-5026.) and maskless (Nimblegen; Singh-Gasson S, Green R D, Yue YJ, Nelson C, Blattner F, Sussman M R, Cerrina F. Maskless fabrication oflight-directed oligonucleotide microarrays using a digital micromirrorarray. Nat. Biotechnol. 1999; 17:974-978) photolithographic deprotectionof photolabile monomers or (iii) digital activation of photogeneratedacids to carry out standard detritylation (Xeotron/Atactic; Gao X L,LeProust E, Zhang H, Srivannavit O, Gulari E, Yu P L, Nishiguchi C,Xiang Q, Zhou X C. A flexible light-directed DNA chip synthesis gated bydeprotection using solution photogenerated acids. Nucleic Acids Res.2001; 29:4744-4750), all of which are herein incorporated by referencein their entirety.

Oligonucleotides made on substrates can be cleaved from their solidsurface and optionally pooled to enable new applications such as, geneassembly, nucleic acid amplification, sequencing libraries, shRNAlibraries etc. (Cleary M A, Kilian K, Wang Y Q, Bradshaw J, Cavet G, GeW, Kulkarni A, Paddison P J, Chang K, Sheth N, et al. Production ofcomplex nucleic acid libraries using highly parallel in situoligonucleotide synthesis. Nature Methods. 2004; 1:241-248), genesynthesis (Richmond K E, Li M H, Rodesch M J, Patel M, Lowe A M, Kim C,Chu L L, Venkataramaian N, Flickinger S F, Kaysen J, et al.Amplification and assembly of chip-eluted DNA (AACED): a method forhigh-throughput gene synthesis. Nucleic Acids Res. 2004; 32:5011-5018;Tian J D, Gong H, Sheng N J, Zhou X C, Gulari E, Gao X L, Church G.Accurate multiplex gene synthesis from programmable DNA microchips.Nature. 2004; 432:1050-1054) and site-directed mutagenesis (Saboulard D,Dugas V, Jaber M, Broutin J, Souteyrand E, Sylvestre J, Delcourt M.High-throughput site-directed mutagenesis using oligonucleotidessynthesized on DNA chips. BioTechniques. 2005; 39:363-368), all of whichare herein incorporated by reference in their entirety.

Successful synthesis of long high-quality oligonucleotides is stronglysupported by high stepwise coupling yields, for example stepwisecoupling yields that are at least about 99.5%. In various embodiments,the methods and compositions of the invention contemplate a couplingyield of more than 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,99.95%, 99.96%, 99.97%, 99.98%, 99.99% or higher. Without being bound bytheory, if the coupling efficiency is lower, e.g. below 99%, the impacton sequence integrity typically follows one of two scenarios. If cappingis used, the low coupling efficiency will be evidenced by short,truncated sequences. If capping is not used, or if capping isunsuccessful, single base deletions will occur in the oligonucleotideand as a consequence, a large number of failure sequences lacking one ortwo nucleotides will be formed. Efficient removal of the 5′-hydroxylprotecting group further supports the synthesis of long, high-qualityoligonucleotides at desirably high yields, such as at very highefficiencies approaching 100% within each cycle, e.g. greater than orequal to 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%,99.96%, 99.97%, 99.98%, 99.99% or higher. This step can be optimizedwith precise control of the dosage of reagents as well as otherenvironmental parameters, using the methods and compositions describedherein, avoiding final product mixtures comprising a family of oligomerswith single base deletions in addition to the desired product.

Further, for synthesis of long oligonucleotides, it is important tominimize the most prevalent side reaction—depurination (Carr P A, Park JS, Lee Y J, Yu T, Zhang S G, Jacobson J M. Protein-mediated errorcorrection for de novo dna synthesis. Nucleic Acids Res. 2004; 32:e162).Depurination results in the formation of an abasic site that typicallydoes not interfere with chain extension. Critical DNA damage occursduring the final nucleobase deprotection under basic conditions, whichalso cleaves oligonucleotide chains at abasic sites. Without being boundby theory, depurination may affect sequence integrity by generatingshort, truncated sequences that can typically be mapped to purinenucleobases. Thus, high yield, high quality synthesis ofoligonucleotides is supported by control of depurination combined withhighly efficient coupling and 5′-hydroxyl deprotection reactions. Withhigh coupling yields and low depurination, long, high qualityoligonucleotides can be synthesized without the need for extensivepurification and/or PCR amplification to compensate for the low yield.The methods and compositions of the invention in various embodimentsprovide conditions to achieve such high coupling yields, lowdepurination, and effective removal of protecting groups.

In various embodiments, the methods and compositions of the inventiondescribed herein rely on standard phosphoramidite chemistry on afunctionalized substrate, e.g. a silylated wafer optionally usingsuitable modifications, known in the art, Typically, after thedeposition of a monomer, e.g. a mononucleotide, a dinucleotide, or alonger oligonucleotide with suitable modifications for phosphoramiditechemistry one or more of the following steps may be performed at leastonce to achieve the step-wise synthesis of high-quality polymers insitu: 1) Coupling, 2) Capping, 3) Oxidation, 4) Sulfurization, 5)Deblocking (detritylation), and 6) Washing. Typically, either oxidationor sulfurization will be used as one of the steps, but not both. FIG. 11exemplifies a four-step phosphoramidite synthesis method comprisingcoupling, capping, oxidation and deblocking steps.

Elongation of a growing oligodeoxynucleotide may be achieved throughsubsequent additions of phosphoramidite building blocks typically viathe formation of a phosphate triester internucleotide bond. During thecoupling step, a solution of phosphoramidite building blocks, e.g.nucleoside phosphoramidite (or a mixture of several phosphoramidites),typically at 0.02-0.2 M concentration, in acetonitrile may be activated,e.g. by a solution of an acidic azole catalyst, 1H-tetrazole,2-ethylthiotetrazole (Sproat et al., 1995, “An efficient method for theisolation and purification of oligoribonucleotides”. Nucleosides &Nucleotides 14 (1&2): 255-273), 2-benzylthiotetrazole (Stutz et al.,2000, “Novel fluoride-labile nucleobase-protecting groups for thesynthesis of 3′(2′)-O-amino-acylated RNA sequences”, Helv. Chim Acta 83(9): 2477-2503; Welz et al., 2002, “5-(Benzylmercapto)-1H-tetrazole asactivator for 2′-O-TBDMS phosphoramidite building blocks in RNAsynthesis”, Tetrahedron Lett., 43 (5): 795-797), 4,5-dicyanoimidazole(Vargeese et al., 1998, “Efficient activation of nucleosidephosphoramidites with 4,5-dicyanoimidazole during oligonucleotidesynthesis”, Nucl. Acids Res., 26 (4): 1046-1050) or a number of similarcompounds, typically at 0.2-0.7 M concentration. The mixing may beachieved in fluid lines of an inkjet while the components are beingdelivered to selected spots of a suitable substrate described in furtherdetail elsewhere herein. The phosphoramidite building blocks, such asthose activated as described above, are typically provided in 1.5, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over thesubstrate-bound material is then brought in contact with the startingsolid support (first coupling) or a support-bound oligonucleotideprecursor (following couplings). In 3′ to 5′ synthesis, 5′-hydroxy groupof the precursor may be set to react with the activated phosphoramiditemoiety of the incoming nucleoside phosphoramidite to form a phosphitetriester linkage. The reaction is also highly sensitive to the presenceof water, particularly when dilute solutions of phosphoramidites areused, and is typically carried out in anhydrous acetonitrile. Upon thecompletion of the coupling, any unbound reagents and by-products may beremoved by a wash step.

The product of the coupling reaction may be treated with a capping agentthat can e.g. esterify failure sequences and/or cleave phosphatereaction products on the heterocyclic bases. The capping step may beperformed by treating the solid support-bound material with a mixture ofacetic anhydride and 1-methylimidazole or DMAP as catalysts and mayserve two purposes: After the completion of the coupling reaction, afraction of the solid support-bound 5′-OH groups (e.g. 0.1 to 1%) mayremain unreacted. These unreacted groups can be permanently blocked fromfurther chain elongation to prevent the formation of oligonucleotideswith an internal base deletion commonly referred to as (n−1) shortmers.The unreacted 5′-hydroxy groups can be acetylated by the cappingmixture. Further, phosphoramidites activated with 1H-tetrazole areunderstood to react, to a small extent, with the O6 position ofguanosine. Without being bound by theory, upon oxidation with I₂/water,this side product, possibly via O6-N7 migration, may undergodepurination. The apurinic sites may end up being cleaved in the courseof the final deprotection of the oligonucleotide thus reducing the yieldof the full-length product. The O6 modifications may be removed bytreatment with the capping reagent prior to oxidation with I₂/water.

The synthesis of oligonucleotide phosphorothioates (OPS; described infurther detail elsewhere herein) typically does not involve theoxidation with I₂/water, and, to that extent, does not suffer from theside reaction described above. On the other hand, the capping mixturemay interfere with the sulfur transfer reaction. Without being bound bytheory, the capping mixture my cause extensive formation of thephosphate triester internucleosidic linkages in place of the desired PStriesters. Therefore, for the synthesis of OPS, the sulfurization stepmay be performed prior to any capping steps.

The support-bound material may be treated with iodine and water,typically in the presence of a weak base (e.g. pyridine, lutidine, orcollidine) to affect oxidization of the phosphite triester into atetracoordinated phosphate triester, a protected precursor of thenaturally occurring phosphate diester internucleosidic linkage.Oxidation may be carried out under anhydrous conditions using, e.g.tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine(CSO). The step of oxidation may be substituted with a sulfurizationstep to obtain oligonucleotide phosphorothioates.

Synthesis of oligonucleotide phosphorothioates (OPS) can be achievedsimilar to that of natural oligonucleotides using the methods andcompositions of the invention in various embodiments. Briefly, theoxidation step can be replaced by the sulfur transfer reaction(sulfurization) and any capping steps can be performed after thesulfurization. Many reagents are capable of the efficient sulfurtransfer, including but not limited to3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

A deblocking (or detrytilation) step may serve to remove blockinggroups, such as the DMT group, e.g. with a solution of an acid, such as2% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA), in aninert solvent (dichloromethane or toluene). A washing step may beperformed. The solid support-bound oligonucleotide precursor is affectedto bear a free 5′-terminal hydroxyl group. Conducting detritylation foran extended time or with stronger than recommended solutions of acidsmay lead to increased depurination of solid support-boundoligonucleotide and thus reduces the yield of the desired full-lengthproduct. Methods and compositions of the invention described hereinprovide for controlled deblocking conditions limiting undesireddepurination reactions.

In some embodiments, an oxidation solution comprising about 0.02 M I₂ inTHF/pyridine/H2O or any suitable variations obvious to one skilled inthe art may be used. The detritylation solution may be 3% dichloroaceticacid (DCA) or 2% tricholoroacetic acid (TCA) in toluene ordichloromethane or any other suitable inert solvent. Suitable variationsof the detrytilation solution are understood to be obvious to oneskilled in the art. The methods and compositions of the invention allowfor the displacement of the detrytilation solution without allowing forsignificant evaporation of the solvent, preventing concentrated spots ofthe depurinating components, e.g. DCA or TCA. For example, a chasingsolution may be run after the detrytilation solution. The density of thechasing solution may be adjusted to achieve a first in first outprocess. A slightly denser chasing solution may be used to achieve thisoutcome. For example, the detrytilation solution may be chased with theoxidation solution. The chasing solution may comprise a quenching agent,such as pyridine. In some embodiments, continuous liquid conditions areused until the deblocking solution is substantially removed from theoligonucleotide synthesis loci on a substrate. The concentration of thedepurinating components may be tightly controlled, e.g. limiting theirvalues on oligonucleotide synthesis loci of a substrate to be less than3-, 2.5-, 2-, 1.5-, 1.4-, 1.3-, 1.25-, 1.2-, 1.15-, 1.1-, 1.05-, 1.04-,1.03-, 1.02-, 1.01-, 1.005-fold or less of the original concentration.

The displacement process can be optimized to adequately control thechemical dosage on the oligonucleotide synthesis loci within a usefulrange. The dosage may collectively refer to the summed kinetic effectsof time, concentration and temperature on both the completion of theintended reaction (detritylation) and the extent of the side reaction(depurination).

Further, detrytilation, by virtue of being reversible, may result in thesynthesis of a series of oligomers lacking one or more of the correctnucleotides. A two-step chemistry proposed by Sierzchala et al.(Solid-phase oligodeoxynucleotide synthesis: A two-step cycle usingperoxy anion deprotection. J. Am. Chem. Soc. 2003; 125:13427-13441) canaddress the issue of depurination by eliminating the use of aciddeprotection of the 5′ or 3′ ends of the growing chain. The two-stepsynthesis cycle makes use of aqueous peroxy anions buffered under mildlybasic conditions, e.g. about pH 9.6, to remove an aryloxycarbonyl group,which substitutes the DMT group commonly used in the four-stepphosphormidite synthesis. Accordingly, the peroxy anion solution, or anysuitable variation with strong nucleophylic and mildly oxidizingproperties permits consolidating deblocking and oxidization steps intoone. Further, high cyclical yields allows for the elimination of cappingsteps.

Deprotection and cleavage of the DNA from the substrate may be performedas described by Cleary et al. (Production of complex nucleic acidlibraries using highly parallel in situ oligonucleotide synthesis.Nature Methods. 2004; 1:241-248), for example by treatment with NH₄OH,by applying ultraviolet light to a photocleavable linker, by targeting,e.g. heat treating, apurinic sites, such as those generated byuracil-DNA glycosylase treatment of incorporated dU-residues, or anysuitable cleavage method known in the art. Oligonucleotides may berecovered after cleavage by lyophylization.

In order to host phosphoramidite chemistry, the surface of theoligonucleotide synthesis loci of a substrate can be chemically modifiedto provide a proper site for the linkage of the growing nucleotide chainto the surface. Various types of surface modification chemistry existwhich allow a nucleotide to attached to the substrate surface. Surfacemodifications may vary in their implementation depending on whether theoligonucleotide chain is to be cleaved from the surface concomitant withdeprotection of the nucleic acid bases, or left attached to the surfaceafter deprotection. Various types of suitable surface modificationchemistries are known in the art and are described atwww.glenresearch.com, which is incorporated herein by reference in itsentirety. One surface modification technique that allows for theexocyclic N atoms of the A, G and C bases to be deprotected while havingthe oligonucleotide chain remain attached to the substrate.

Another scheme comprises reacting a trialkoxysilyl amine (e.g.(CH3CH2O)3Si-(CH2)2-NH2) with the glass or silica surface SiOH groups,followed by reaction with succinic anhydride with the amine to createand amide linkage and a free OH on which the nucleotide chain growthcould commence.

A third type of linker group may be based on photocleavable primers.This type of linker allows for oligonucleotide to be removed from thesubstrate (by irradiation with light, e.g. ^(˜)350 nm light) withoutcleaving the protecting groups on the nitrogenous functionalities oneach base. The typical ammonia or NH3 treatment deprotects everythingwhen used as the reagent to cleave the oligomers from the substrate. Theuse of photocleavable linkers of this sort is described atwww.glenresearch.com. Various other suitable cleavable linker groups areknown in the art and may alternatively be used.

Time frames for oxidation and detritylation may typically be about 30 sand 60 s, respectively. The reagents may be drained, followed by washesof acetonitrile (ACN). In the depurination controlled detritylationprocesses, the detritylation solution may be driven out using acontinuous inflow of oxidation solution without a drain step in between.

Precise control of the flow of reagents during the in situ synthesissteps allows for improved yield, uniformity and quality of the products.For example, the acid concentration and detritylation times can beprecisely controlled. A water contact angle for the substrate, inparticular, for regions of in situ synthesis and/or surrounding areas,may be chosen in order to reduce depurination and/or speed of synthesis.Proper desired values of water contact angle are described elsewhereherein. In some embodiments, lower amount of depurination may beachieved on surfaces of higher surface energy, i.e. lower contact angle.

The methods and compositions of the invention allow for a reduced rateof depurination during oligonucleotide synthesis, e.g. at a rate of lessthan 0.1%, 0.09%, %, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%,0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%,0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%,0.0002%, 0.0001% per cycle or less. Further, methods and compositions ofthe invention described herein allow for the reduction or elimination ofa depurination gradient across the surface of a substrate supporting insitu synthesis of oligonucleotides. Thus, highly uniform, high quality,and high-yield oligonucleotide synthesis can be achieved on substratesthat can host a high density of resolved oligonucleotide loci.

In situ synthesis of oligonucleotides typically starts with the solidsupport being relatively hydrophobic, and subsequently growingincreasingly more hydrophylic with the synthesis of oligonucleotidefeatures affecting its surface energy. Oligonucleotide features can gainsubstantial surface energy with increasing oligonucleotide length.Generally, these sites or features consisting of protectedoligonucleotide acquire enough surface energy to become spontaneouslywetting to high surface tension organic solvents commonly used inphosphoramidite synthesis, such as acetonitrile or propylene carbonate,after about 10-20 synthesis cycles. The methods and compositions of theinvention allow for varying parameters, such as time, flow rate,temperature, volume, viscosity, or reagent concentration, during thesynthesis of a growing oligonucleotide as a function of length toaccount for the changing surface properties on loci of oligonucleotidesynthesis. Such a variation may be applied by continuously changingparameters in constant or varying increments at repeating cycles of thesynthesis. Alternatively, parameters may be changed at only selectedcycles of the synthesis and can optionally follow a pattern, such asevery other cycle, every third, fourth, fifth, sixth, seventh, eighth,ninth, tenth cycle etc.

In various embodiments, the methods and compositions of the inventioncontemplate a library of oligonucleotides synthesized on a substrate,wherein the library comprises oligonucleotides of varying sizes, asdescribed in further detail elsewhere herein. Further, the methods andcompositions of the invention allow for the synthesis of substantiallysimilar amounts of oligonucleotides, or in some cases varyingpreselected amounts of oligonucleotides, of varying size, sequence ornucleotide composition on a substrate. The variation in amounts may belimited to less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%,0.5%, 0.1% or less between any two synthesized oligonucleotides, oralternatively, as relative error or percent deviation across thelibrary. The methods and compositions of the invention described hereincontemplate synthesized oligonucleotides on a substrate at desiredamounts as described in further detail elsewhere herein.

In some embodiments, the methods and compositions of the inventionpermit the synthesis of libraries of oligonucleotides on substrates, inwhich the stoichiometry of each oligonucleotide is tightly controlledand tunable by varying the relative number of features synthesized.Suitable surface functionalizations and coatings to finetune the densityof growing oligonucleotides on resolved loci of substrates are describedin further detail elsewhere herein and can be uniformly applied to allmicrostructures of a substrate, or alternatively, can be applied atselected amounts and ratios to individual microstructures.

The in situ synthesis methods include those described in U.S. Pat. No.5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 andthe references cited therein for synthesizing polynucleotides(specifically, DNA) using phosphoramidite or other chemistry. Additionalpatents describing in situ nucleic acid array synthesis protocols anddevices include U.S. Pub. No. 2013/0130321 and U.S. Pub. No.2013/0017977, and the references cited therein, incorporated herein byreference in their entirety.

Such in situ synthesis methods can be basically regarded as iteratingthe sequence of depositing droplets of: a protected monomer ontopredetermined locations on a substrate to link with either a suitablyactivated substrate surface (or with previously deposited deprotectedmonomer); deprotecting the deposited monomer so that it can react with asubsequently deposited protected monomer; and depositing anotherprotected monomer for linking. Different monomers may be deposited atdifferent regions on the substrate during any one cycle so that thedifferent regions of the completed array will carry the differentbiopolymer sequences as desired in the completed array. One or moreintermediate further steps may be required in each iteration, such asoxidation, sulfurization, and/or washing steps.

Various methods which can be used to generate an array ofoligonucleotides on a single substrate are described in U.S. Pat. Nos.5,677,195, 5,384,261, and in PCT Publication No. WO 93/09668. In themethods disclosed in these applications, reagents are delivered to thesubstrate by either (1) flowing within a channel defined on predefinedregions or (2)“spotting” on predefined regions, or (3) through the useof photoresist. However, other approaches, as well as combinations ofspotting and flowing, can be employed. In each instance, certainactivated regions of the substrate are mechanically separated from otherregions when the monomer solutions are delivered to the various reactionsites. Thus, in situ synthesis of oligonucleotides can be achievedapplying various suitable methods of synthesis known in the art to themethods and compositions described herein. One such method is based on aphotolithographic technique which involves direct in situ synthesis ofoligonucleotides at resolved pre-determined sites on the solid orpolymeric surface, using photolabile protecting groups (Kumar et al.,2003). The hydroxyl groups can be generated on the surface and blockedby photolabile-protecting groups. When the surface is exposed to ˜UVlight, e.g. through a photolithographic mask, a pattern of free hydroxylgroups on the surface may be generated. These hydroxyl groups can reactwith photoprotected nucleosidephosphoramidites, according tophosphoramidite chemistry. A second photolithographic mask can beapplied and the surface can be exposed to UV light to generate secondpattern of hydroxyl groups, followed by coupling with 5′-photoprotectednucleosidephosphoramidite. Likewise, patterns can be generated andoligomer chains can be extended. Several photolabile-protecting groups,which can be removed cleanly and rapidly from the 5′-hydroxylfunctionalities are known in the art. Without being bound by theory, thelability of a photocleavable group depends on the wavelength andpolarity of a solvent employed and the rate of photocleavage may beaffected by the duration of exposure and the intensity of light. Thismethod can leverage a number of factors, e.g. accuracy in alignment ofthe masks, efficiency of removal of photo-protecting groups, and theyields of the phosphoramidite coupling step. Further, unintended leakageof light into neighboring sites can be minimized. The density ofsynthesized oligomer per spot can be monitored by adjusting loading ofthe leader nucleoside on the surface of synthesis.

It is understood that the methods and compositions of the invention canmake use of a number of suitable techniques of construction that arewell known in the art e.g., maskless array synthesizers, light directedmethods utilizing masks, flow channel methods, spotting methods etc. Insome embodiments, construction and/or selection oligonucleotides may besynthesized on a solid support using maskless array synthesizer (MAS).Maskless array synthesizers are described, for example, in PCTapplication No. WO 99/42813 and in corresponding U.S. Pat. No.6,375,903. Other examples are known of maskless instruments which canfabricate a custom DNA microarray in which each of the features in thearray has a single-stranded DNA molecule of desired sequence. Othermethods for synthesizing construction and/or selection oligonucleotidesinclude, for example, light-directed methods utilizing masks, flowchannel methods, spotting methods, pin-based methods, and methodsutilizing multiple supports. Light directed methods utilizing masks(e.g., VLSIPS™ methods) for the synthesis of oligonucleotides isdescribed, for example, in U.S. Pat. Nos. 5,143,854, 5,510,270 and5,527,681. These methods involve activating predefined regions of asolid support and then contacting the support with a preselected monomersolution. Selected regions can be activated by irradiation with a lightsource through a mask much in the manner of photolithography techniquesused in integrated circuit fabrication. Other regions of the supportremain inactive because illumination is blocked by the mask and theyremain chemically protected. Thus, a light pattern defines which regionsof the support react with a given monomer. By repeatedly activatingdifferent sets of predefined regions and contacting different monomersolutions with the support, a diverse array of polymers is produced onthe support. Other steps, such as washing unreacted monomer solutionfrom the support, can be optionally used. Other applicable methodsinclude mechanical techniques such as those described in U.S. Pat. No.5,384,261. Additional methods applicable to synthesis of constructionand/or selection oligonucleotides on a single support are described, forexample, in U.S. Pat. No. 5,384,261. For example, reagents may bedelivered to the support by flowing within a channel defined onpredefined regions or “spotting” on predefined regions. Otherapproaches, as well as combinations of spotting and flowing, may beemployed as well. In each instance, certain activated regions of thesupport are mechanically separated from other regions when the monomersolutions are delivered to the various reaction sites. Flow channelmethods involve, for example, microfluidic systems to control synthesisof oligonucleotides on a solid support. For example, diverse polymersequences may be synthesized at selected regions of a solid support byforming flow channels on or in a surface of the support through whichappropriate reagents flow or in which appropriate reagents are placed.Spotting methods for preparation of oligonucleotides on a solid supportinvolve delivering reactants in relatively small quantities by directlydepositing them in selected regions or structures fluidically connectedto the same. In some steps, the entire support surface can be sprayed orotherwise coated with a solution. Precisely measured aliquots of monomersolutions may be deposited dropwise by a dispenser that moves fromregion to region. Pin-based methods for synthesis of oligonucleotides ona solid support are described, for example, in U.S. Pat. No. 5,288,514.Pin-based methods utilize a support having a plurality of pins or otherextensions. The pins are each inserted simultaneously into individualreagent containers in a tray.

In an alternative approach, light directed synthesis of high densitymicroarrays can be achieved in 5′-3′ direction (Albert et al., 2003).This approach allows for downstream reactions, such as parallelgenotyping or sequencing, to be done on the synthesis surface, because3′-end is available for enzymatic reactions, such as sequence specificprimer extension and ligation reactions. Complete or substantiallycomplete deprotection of photoprotected 5′-OH groups, base-assistedphoto-deprotection of NPPOC (2-(2-nitrophenyl) propoxy carbonyl) can beused (Beier et al., 2002).

The methods and compositions described herein may facilitate theproduction of synthetic nucleic acids using in situ synthesis onsubstrates of various geometries, including planar or irregularsurfaces. Various materials suitable for these substrates, e.g. silicon,are described herein are otherwise known in the art. A substrate may beloaded with a multiplicity of different sequences during the synthesis.In situ synthesis methods on substrates allows for the preparation of amultiplicity of oligomers of different and defined sequences ataddressable locations on a common support. The methods and compositionsdescribed herein allow for the in situ synthesis of oligonucleotidesthat are longer and higher quality as further described elsewhereherein. The synthesis steps can incorporate various sets of feedmaterials, in the case of oligonucleotide synthesis, as a rule the 4bases A, G, T and C, as well as suitable modified bases known in the artsome of which are described herein, may be used building up desiredsequences of nucleic acid polymers in a resolved manner on a support orsubstrate.

The fabrication and application of high density oligonucleotides onsolid support, e.g. arrays, have been further disclosed previously in,for example, PCT Publication No's WO 97/10365, WO 92/10588, U.S. Pat.No. 6,309,822 filed Dec. 23, 1996; U.S. Pat. No. 6,040,138 filed on Sep.15, 1995; Ser. No. 08/168,904 filed Dec. 15, 1993; Ser. No. 07/624,114filed on Dec. 6, 1990, Ser. No. 07/362,901 filed Jun. 7, 1990, and inU.S. Pat. No. 5,677,195, all incorporated herein for all purposes byreference. In some embodiments using high density arrays, high densityoligonucleotide arrays are synthesized using methods such as the VeryLarge Scale Immobilized Polymer Synthesis (VLSIPS) disclosed in U.S.Pat. Nos. 5,445,934 and 6,566,495, both incorporated herein for allpurposes by reference. Each oligonucleotide occupies a known location ona substrate.

Various other suitable methods of forming high density arrays ofoligonucleotides, peptides and other polymer sequences with a minimalnumber of synthetic steps are known in the art. The oligonucleotideanalogue array can be synthesized on a solid substrate by a variety ofmethods, including, but not limited to, light-directed chemical couplingand mechanically directed coupling. See Pirrung et al., U.S. Pat. No.5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al.,PCT Publication Nos. WO 92/10092 and WO 93/09668 and U.S. Ser. No.07/980,523, which disclose methods of forming vast arrays of peptides,oligonucleotides and other molecules using, for example, light-directedsynthesis techniques. See also, Fodor et al., Science, 251, 767-77(1991). These procedures for synthesis of polymer arrays are nowreferred to as VLSIPS procedures. Using the VLSIPS approach, oneheterogeneous array of polymers is converted, through simultaneouscoupling at a number of reaction sites, into a different heterogeneousarray. See, U.S. application Ser. Nos. 07/796,243 and 07/980,523.

In the event that an oligonucleotide analogue with a polyamide backboneis used in the VLSIPS procedure, it is often unsuitable to usephosphoramidite chemistry to perform the synthetic steps, since themonomers do not attach to one another via a phosphate linkage. Instead,peptide synthetic methods can be substituted e.g., as described byPirrung et al. in U.S. Pat. No. 5,143,854, which is herein incorporatedby reference in its entirety.

The individual molecular species can be demarcated by separate fluidiccompartments for addition of the synthesis feed materials, as is thecase e.g. in the so-called in situ spotting method or piezoelectrictechniques, based on inkjet printing technology (A. Blanchard, inGenetic Engineering, Principles and Methods, Vol. 20, Ed. J. Sedlow,111-124, Plenum Press; A. P. Blanchard, R. J. Kaiser, L. E. Hood,High-Density Oligonucleotide Arrays, Biosens. & Bioelectronics 11, 687,1996). Resolved in situ synthesis of oligonucleotides can further beachieved by the spatially-resolved activation of synthesis sites, whichis possible through selective illumination, through selective orspatially-resolved generation of activation reagents (deprotectionreagents) or through selective addition of activation reagents(deprotection reagents).

Examples of the methods known to date for the in situ synthesis ofarrays are photolithographic light-based synthesis (McGall, G. et al.;J. Amer. Chem. Soc. 119; 5081-5090; 1997), projector-based light-basedsynthesis (PCT/EP99/06317), fluidic synthesis by means of physicalseparation of the reaction spaces (known by a person skilled in the artfrom the work of Prof. E. Southern, Oxford, UK, and of the companyOxford Gene Technologies, Oxford, UK), indirect projector-basedlight-controlled synthesis by light-activated photo-acids and suitablereaction chambers or physically separated reaction spaces in a reactionsupport, electronically induced synthesis by spatially-resolveddeprotection on individual electrodes on the support using protonproduction induced by the electrodes, and fluidic synthesis byspatially-resolved deposition of the activated synthesis monomers (knownfrom A. Blanchard, in Genetic Engineering, Principles and Methods, Vol.20, Ed. J. Sedlow, 111-124, Plenum Press; A. P. Blanchard, R. J. Kaiser,L. E. Hood, High-Density Oligonucleotide Arrays, Biosens. &Bioelectronics 11, 687, 1996).

Methods of preparation of synthetic nucleic acids, in particular nucleicacid double strands on a common solid support, are also known from PCTPublications WO 00/49142 and WO 2005/051970, both of which are hereinincorporated by reference in their entirety.

In situ preparation of nucleic acid arrays, can be achieved, 3′ to 5′,as well as the more traditional 5′ to 3′ direction. Addition of reagentsmay be achieved by pulse-jet depositing, e.g. an appropriate nucleotidephosphoramidite and an activator to each resolved locus on or in asubstrate surface, e.g., a coated silicon wafer surface. The resolvedloci of the substrate may further be subjected to additional reagents ofthe other phosphoramidite cycle steps (deprotection of the 5′-hydroxylgroup, oxidation, sulfurization and/or sulfurization), which may beperformed in parallel. The deposition and common phosphoramidite cyclesteps may be performed without moving the oligonucleotide synthesiswafer. For example, the reagents may be passed over resolved loci withina substrate, by flowing them through the substrate from one surface tothe opposite surface of the substrate. Alternatively, the substrate maybe moved, e.g. to a flow cell, for some of the phosphoramidite cyclesteps. The substrate can then be repositioned, re-registered, and/orre-aligned before printing a next layer of monomers.

Substrates with oligonucleotides can be fabricated using drop depositionfrom pulsejets of either polynucleotide precursor units (such asmonomers) in the case of in situ fabrication, or a previouslysynthesized polynucleotide. Such methods are described in detail in, forexample, the U.S. Pub. No. 2013/0130321 and U.S. Pub. No. 2013/0017977,and the references cited therein, incorporated herein by reference intheir entirety. These references are incorporated herein by reference.Other drop deposition methods can be used for fabrication, as describedelsewhere herein. Also, instead of drop deposition methods, lightdirected fabrication methods may be used, as are known in the art.Interfeature areas need not be present particularly when the arrays aremade by light directed synthesis protocols.

A variety of known in situ fabrication devices can be adapted, whererepresentative pulse-jet devices include, but are not limited to, thosedescribed in U.S. Pub. No. US2010/0256017, U.S. Pat. Pub. No.US20120050411, and U.S. Pat. No. 6,446,682, the disclosures of whichpatents are herein incorporated by reference in their entirety.

In various embodiments, biopolymer arrays on or inside substrates can befabricated using either deposition of the previously obtainedbiopolymers or in situ synthesis methods. The deposition methodstypically involve depositing biopolymers at predetermined locations onor in a substrate which are suitably activated such that the biopolymerscan link thereto. Biopolymers of different sequences may be deposited atdifferent regions on or in a substrate. Typical procedures known in theart for deposition of previously obtained polynucleotides, particularlyDNA, such as whole oligomers or cDNA, includes, but is not limited toloading the polynucleotide into a drop dispenser in the form of a pulsejet head and fired onto the substrate. Such a technique has beendescribed in WO 95/25116 and WO 98/41531, both of which are hereinincorporated by reference in their entirety. Various suitable forms ofinkjets for drop depositions to resolved sites of a substrate are knownin the art.

In some embodiments, the invention may rely on the use ofpre-synthesized oligonucleotides within an entire oligonucleotidelibrary or parts thereof, for example, an oligonucleotide libraryimmobilized on a surface. Substrates supporting a high density ofnucleic acid arrays can be fabricated by depositing presynthesized ornatural nucleic acids in predetermined positions on, in, or through asubstrate. Synthesized or natural nucleic acids may be deposited onspecific locations of a substrate by light directed targeting,oligonucleotide directed targeting, or any other suitable method knownin the art. Nucleic acids can also be directed to specific locations. Adispenser that moves from region to region to deposit nucleic acids inspecific spots can be used. The dispenser may deposit the nucleic acidthrough microchannels leading to selected regions. Typical dispensersinclude a micropipette or capillary pin to deliver nucleic acid to thesubstrate and a robotic system to control the position of themicropipette with respect to the substrate. In other embodiments, thedispenser includes a series of tubes, a manifold, an array of pipettesor capillary pins, or the like so that various reagents can be deliveredto the reaction regions simultaneously.

Attachment of pre-synthesized oligonucleotide and/or polynucleotidesequences to a support and in situ synthesis of the same usinglight-directed methods, flow channel and spotting methods, inkjetmethods, pin-based methods and bead-based methods are further set forthin the following references: McGall et al. (1996) Proc. Natl. Acad. Sci.U.S.A. 93: 13555; Synthetic DNA Arrays In Genetic Engineering, Vol. 20:111, Plenum Press (1998); Duggan et al. (1999) Nat. Genet. S21: 10;Micro arrays: Making Them and Using Them In Microarray Bioinformatics,Cambridge University Press, 2003; U.S. Patent Application PublicationNos. 2003/0068633 and 2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890,6,824,866, 6,800,439, 6,375,903 and 5,700,637; and PCT Publication Nos.WO 04/031399, WO 04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO03/065038, WO 03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO03/040410 and WO 02/24597; the disclosures of which are incorporatedherein by reference in their entirety for all purposes. In someembodiments, pre-synthesized oligonucleotides are attached to a supportor are synthesized using a spotting methodology wherein monomerssolutions are deposited dropwise by a dispenser that moves from regionto region (e.g., inkjet). In some embodiments, oligonucleotides arespotted on a support using, for example, a mechanical wave actuateddispenser.

The systems described herein can further include a member for providinga droplet to a first spot (or feature) having a plurality ofsupport-bound oligonucleotides. In some embodiments, the droplet caninclude one or more compositions comprising nucleotides oroligonucleotides (also referred herein as nucleotide additionconstructs) having a specific or predetermined nucleotide to be addedand/or reagents that allow one or more of hybridizing, denaturing, chainextension reaction, ligation, and digestion. In some embodiments,different compositions or different nucleotide addition constructs maybe deposited at different addresses on the support during any oneiteration so as to generate an array of predetermined oligonucleotidesequences (the different features of the support having differentpredetermined oligonucleotide sequences). One particularly useful way ofdepositing the compositions is by depositing one or more droplet, eachdroplet containing the desired reagent (e.g. nucleotide additionconstruct) from a pulse jet device spaced apart from the supportsurface, onto the support surface or features built into the supportsurface.

To make it possible to automate the chemical method of polymer synthesisfrom subunits, solid phases are often employed, on which the growingmolecular chain is anchored. On completion of synthesis it may be splitoff, which may be achieved by breaking a suitable linker between theactual polymer and the solid phase. For automation, the method mayemploy a substrate surface directly or the method may employ a substratesurface of solid phases in the form of activated particles, which arepacked in a column or microchannel in a substrate, e.g. controlled poreglass (CPG). The substrate surface at times can carry one specificallyremovable type of oligo with a predetermined sequence. The individualsynthesis reagents can be then added in a controllable manner. Thequantity of molecules synthesized can be controlled by various factors,including but not limited to the size of the dedicated substratesurface, amount of support material, the size of the reaction batches,available functionalized substrate area for synthesis, the degree offunctionalization, or the duration of the synthesis reaction.

Thus, various embodiments of the invention relate to the manufacturingand use of substrates holding a library of compositions, typicallyoligonucleotides. A substrate with resolved features is “addressable”when it has multiple regions of different moieties (e.g., differentpolynucleotide sequences) such that a region (i.e., a “feature” or“spot” of the substrate) at a particular predetermined location (i.e.,an “address”) on the substrate will detect a particular target or classof targets (although a feature may incidentally detect non-targets ofthat location). Substrate features are typically, but need not be,separated by intervening spaces. In some cases, features may be builtinto a substrate and may create one-, two-, or three-dimensionalmicrofluidic geometries. A “substrate layout” refers to one or morecharacteristics of the features, such as feature positioning on thesubstrate, one or more feature dimensions, and an indication of a moietyat a given location.

Synthesis of Other Molecules

The subject methods and compositions can be used to synthesize othertypes of molecules of interest. The synthesis of peptides at selectedgrid regions is one such case. Various suitable chemistries used instepwise growth of peptides on an array surface are known in the art.The peptide synthesis techniques described in U.S. Pat. No. 5,449,754,incorporated herein by reference in its entirety, may be used with thepresent invention. The methods and compositions of the inventiondescribed herein also find uses in chemical synthesis of drugs, proteininhibitors or any chemical synthesis in which the rapid synthesis of aplurality of compounds is desired.

Gene Assembly

In various embodiments, the present invention relates to the preparationof a polynucleotide sequence (also called “gene”) using assembly ofoverlapping shorter oligonucleotides synthesized or spotted on substratesurfaces or alternatively, substrates housing suitable surfaces for thesynthesis or spotting of oligonucleotides, e.g. beads. The shorteroligonucleotides may be patchworked together on the same strand usingannealing oligonucleotides with complementary regions to consecutiveassembled oligonucleotides, e.g. using a polymerase lacking stranddisplacement activity, a ligase, Click chemistry, or any other suitableassembly method known in the art. In this fashion, the sequence of theannealing nucleotide may be replicated between the consecutiveoligonucleotides of the opposing strand. In some cases, consecutiveoligonucleotides of the same strand may be stitched together without theintroduction of sequence elements from the annealing oligonucleotide,for example using a ligase, Click chemistry, or any other suitableassembly chemistry known in the art. In some cases, longerpolynucleotides can be synthesized hierarchically through rounds ofassembly involving shorter polynucleotides/oligonucleotides.

Genes or genomes can be synthesized de novo from oligonucleotides byassembling large polynucleotides as described in the synthesis of aviral genome (7.5 kb; Cello et al, Science, 2002, 297, 1016),bacteriophage genome (5.4 kb; Smith et al, Proc. Natl. Acad. Sci. USA,2003, 100, 15440), and a gene cluster as large as 32 kb (Kodumal et al,Proc. Natl. Acad. Sci. USA, 2004, 101, 15573), all of which are hereinincorporated by reference in their entirety. Libraries of long syntheticDNA sequence can be manufactured, following the methods described in the582 kb the genome assembly of a bacterium (Mycoplasma genitalium) byVenter and co-workers (Gibson et al, Science, 2008, 319, 1215), which isincorporated herein by reference in its entirety. Furthermore, large DNAbiomolecules can be constructed with oligonucleotides, as described forthe case of a 15 kb nucleic acid (Tian et al, Nature, 2004, 432, 1050;incorporated herein by reference in its entirety). The methods andcompositions of the invention contemplate large libraries of de novosynthesized polynucleotide sequences using gene assembly methodsdescribed herein or known in the art. The synthesis of such sequencesare typically performed in parallel in high densities on suitableregions of substrates that are described in further detail elsewhereherein. Further, these large libraries may be synthesized with very lowerror rates, described in further detail elsewhere herein.

Genes may be assembled from large numbers of synthesizedoligonucleotides that are pooled. For example, gene synthesis using apool of 600 distinct oligonucleotides can be applied as described byTian et al. (Tian et al. Nature, 432:1050, 2004). The length of theassembled genes can be further extended by using longeroligonucleotides. For even larger genes and DNA fragments, for examplelarger than about 0.5, 1, 1.5, 2, 3, 4, 5 kb, or more, more than onerounds of synthesis may be applied, typically within a hierarchical geneassembly process. PCR assembly and synthesis from oligonucleotides asdisclosed herein may be adapted for use in series, as described below.

A variety of gene assembly methods can be used according to the methodsand compositions of the invention, ranging from methods such asligase-chain reaction (LCR) (Chalmers and Curnow, Biotechniques, 30(2),249-52, 2001; Wosnick et al, Gene, 60(1), 115-27, 1987) to suites of PCRstrategies (Stemmer et al, 164, Gene, 49-53, 1995; Prodromou and L. H.Pearl, 5(8), Protein Engineering, 827-9, 1992; Sandhu et al, 12(1),BioTechniques, 14-6, 1992; Young and Dong, Nucleic Acids Research,32(7), e59, 2004; Gao et al, Nucleic Acids Res., 31, e143, 2003; Xionget al, Nucleic Acids Research, 32(12), e98, 2004) (FIG. 11). While mostassembly protocols start with pools of overlapping synthesized oligosand end with PCR amplification of the assembled gene, the pathwaybetween those two points can be quite different. In the case of LCR, theinitial oligo population has phosphorylated 5′ ends that allow a ligase,e.g. Pfu DNA ligase, to covalently connect these “building blocks”together to form the initial template. PCR assembly, however, typicallymakes use of unphosphorylated oligos, which undergo repetitive PCRcycling to extend and create a full length template. Additionally, theLCR processes may require oligo concentrations in the μM range, whereasboth single stranded and double stranded PCR options have concentrationrequirements that are much lower (e.g. nM range).

Published synthesis attempts have used oligos ranging in size from 20-70bp, assembling through hybridization of overlaps (6-40 bp). Since manyfactors are determined by the length and composition of oligos (Tm,secondary structure, etc.), the size and heterogeneity of thispopulation could have a large effect on the efficiency of assembly andquality of assembled genes. The percentage of correct final DNA productrelies on the quality and number of “building block” oligos. Shorteroligos have lower mutated rate compared with that of longer oligos, butmore oligos are required to build the DNA product. In addition, thereduced overlaps of shorter oligos results in lower T_(m) of theannealing reaction, which promotes non-specific annealing, and reducethe efficiency of the assembly process. Methods and compositions of theinvention address this problem by delivering long oligonucleotides withlow error rates.

A time varying thermal field refers to the time regulated heating of themicrofluidic device to allow PCR amplification or PCA reactions tooccur. The time varying thermal field may be applied externally, forexample by placing a device substrate with reactors, e.g. nanoreactorson top of a thermal heating block, or integrated within a microfluidicdevice, for example as a thin film heater located immediately below thePCA and PCR chambers. A temperature controller can vary the temperatureof the heating element in conjunction with a temperature sensor linkedto a heater element, or integrated into the reaction chamber. A timercan vary the duration of heat applied to the reaction chambers.

The temperature of the thermal field may vary according to thedenaturation, annealing and extension steps of PCR or PCA reactions.Typically, nucleic acids are denatured at about 95° C. for 2 min,followed by 30 or more cycles of denaturation at 95° C. for 30 sec,annealing at 40-60° C. for 30 sec and extension at about 72° C. for 30sec, and a last extension of 72° C. for 10 min. The duration andtemperatures used may vary depending on the composition of theoligonucleotides, PCR primers, amplified product size, template, and thereagents used, for example the polymerase.

Polymerases are enzymes that incorporate nucleoside triphosphates, ordeoxynucleoside triphosphates, to extend a 3′ hydroxyl terminus of a PCRprimer, an oligonucleotide or a DNA fragment. For a general discussionconcerning polymerases, see Watson, J. D. et al, (1987) MolecularBiology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif.Suitable polymerases include, but are not limited to, KOD polymerase;Pfu polymerase; Taq-polymerase; E. coli DNA polymerase I, “Klenow”fragment, T7 polymerase, T4 polymerase, T5 polymerase and reversetranscriptase, all of which are known in the art. A polymerase havingproof-reading capability, such as Pfu and Pyrobest may be used toreplicate DNA with high fidelity. Pfu DNA polymerase possesses 3′ to 5′exonuclease proof-reading activity, thus it may correct nucleotidemisincorporation errors. In various embodiments of the invention, thenucleic acid fragments are joined together preferably by a specifichybridization reaction between overlapping regions of mutuallycomplementary segments of the nucleic acid fragments, thereby obtaininglonger synthetic double-stranded nucleic acids. The individual sequencesegments used for building up longer nucleic acids can have a length of,e.g. 20-200, 50-300, 75-350 or 100-400 nucleotide building blocks. Thoseof skill in the art appreciate that the sequence segment length may fallwithin any range bounded by any of these values (e.g., 20-350 or200-350).

The sequence segments are preferably selected in such a way that they atleast partially overlap a sequence segment of the antisense strand ofthe complementary nucleic acid that is to be synthesized, so that thenucleic acid strand to be synthesized can be built up by hybridizationof individual sequence segments. In an alternative embodiment, thesequence segments are preferably selected so that the sequence segmentson both strands of the nucleic acid to be synthesized completelyoverlap, and accordingly preparation of a more or less complete doublestrand now only requires covalent linkage of the phosphodiesterbackbone. The length of the complementary regions or overlaps betweenindividual fragments can be e.g. 10-50, 10-100, 12-25, 20-80, 15-20, or15-25 nucleotide building blocks. Those of skill in the art appreciatethat the sequence segment length may fall within any range bounded byany of these values (e.g., 25-100 or 10-25). If the overlapping orcomplementarity region between two nucleic acid fragments has a high ATcontent, e.g. an AT content of greater than 50%, 60%, 65%, or higher thebinding constant is lower in comparison with GC-richer sequences.Accordingly, for thermodynamic reasons, hybridization between thesefragments may be of comparatively low efficiency. This can have aninfluence on the assembly of 2 or more fragments. A possiblesequence-dependent consequence is a reduced yield of nucleic acid doublestrands with the correct target sequence. Accordingly, sequence segmentsto assemble genes can be designed with desired levels of GC content inoverlapping regions, for example GC content of more than 35, 40, 45, 50,55, 60, 65%, or higher. A more detailed discussion of exemplary geneassembly methods can be found in U.S. Pat. No. 8,367,335, which isherein incorporated by reference in its entirety.

In various embodiments, polymerase chain reaction (PCR)-based andnon-polymerase-cycling-assembly (PCA)-based strategies can be used forchemical gene synthesis. In addition, non-PCA-based chemical genesynthesis using different strategies and methods, including enzymaticgene synthesis, annealing and ligation reaction, simultaneous synthesisof two genes via a hybrid gene, shotgun ligation and co-ligation,insertion gene synthesis, gene synthesis via one strand of DNA,template-directed ligation, ligase chain reaction, microarray-mediatedgene synthesis, Blue Heron solid support technology, Sloning buildingblock technology, RNA-mediated gene assembly, the PCR-basedthermodynamically balanced inside-out (TBIO) (Gao et al., 2003),two-step total gene synthesis method that combines dual asymmetrical PCR(DA-PCR) (Sandhu et al., 1992), overlap extension PCR (Young and Dong,2004), PCR-based two-step DNA synthesis (PTDS) (Xiong et al., 2004b),successive PCR method (Xiong et al., 2005, 2006a), or any other suitablemethod known in the art can be used in connection with the methods andcompositions described herein, for the assembly of longerpolynucleotides from shorter oligonucleotides.

The DNA sequences that have been chemically synthesized using themethods and compositions of the invention may extend to longpolynucleotide sequences, for example, polynucleotide sequences of morethan 500, 750, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 4000, 5000,6000, 7500, 10000, 20000, 30000, 40000, 50000, 75000, 100000 base pairsor longer. The methods and compositions of the invention also allow forchemically synthesized polynucleotide sequences with very low errorrates, as further described elsewhere herein.

In various embodiments, variations of the polymerase-mediated assemblytechniques, collectively termed polymerase construction andamplification, are used for chemical synthesis of polynucleotides. Someof the commonly used technologies known in the art for custom genesynthesis are based on polymerase cycling assembly and may achieve denovo synthesis of longer polynucleotides through the assembly of a poolof oligonucleotides. The pool of oligonucleotides may be synthesized asbuilding blocks for use in various gene synthesis techniques. Thesequence, length and precise distribution of the oligonucleotides, aswell as any sequence overlaps within the pool, may be designed accordingto the desired polynucleotide sequence and the assembly method used. Thedesired full-length DNA may be obtained, for example, by a few steps ofPCR with necessary temperature conditions for denaturing, annealing, andelongating overlapping oligonucleotides.

PCR Assembly (PCA)

PCR assembly uses polymerase-mediated chain extension in combinationwith at least two oligonucleotides having complementary ends which cananneal such that at least one of the polynucleotides has a free3′-hydroxyl capable of polynucleotide chain elongation by a polymerase(e.g., a thermostable polymerase such as Taq polymerase, VENT™polymerase (New England Biolabs), KOD (Novagen) and the like).Overlapping oligonucleotides may be mixed in a standard PCR reactioncontaining dNTPs, a polymerase, and buffer. The overlapping ends of theoligonucleotides, upon annealing, create regions of double-strandednucleic acid sequences that serve as primers for the elongation bypolymerase in a PCR reaction. Products of the elongation reaction serveas substrates for formation of a longer double-strand nucleic acidsequences, eventually resulting in the synthesis of full-length targetsequence. The PCR conditions may be optimized to increase the yield ofthe target long DNA sequence.

Various PCR based methods can be used to synthesize genes fromoligonucleotides. These methods include, but are not limited to, thethermodynamically balanced inside-out (TBIO) method (Gao et al, NucleicAcids Research, 31:e143, 2003), successive PCR (Xiong et al, NucleicAcids Research, 32:e98, 2004), dual asymmetrical PCR (DA-PCR) (Sandhu etal, Biotechniques, 12:14, 1992), overlap extension PCR (OE-PCR) (Youngand Dong, Nucleic Acids Research, 32:e59, 2004; Prodromou and Pearl,Protein Eng., 5:827, 1992) and PCR-based two step DNA synthesis (PTDS)(Xiong et al, Nucleic Acids Research, 32:e98, 2004), all of which areincorporated by reference herein in their entirety and can be adapted toassemble a PCR template in a microfluidic device.

DA-PCR is a one-step process for constructing synthetic genes. In oneexample, four adjacent oligonucleotides of, e.g. 17-100 bases in lengthwith overlaps of, e.g. 15-17 bases are used as primers in a PCRreaction. Other suitable oligonucleotide and overlap sizes are withinthe bounds of the invention as further described herein. The quantity ofthe two internal primers is highly limited, and the resultant reactioncauses an asymmetric single-stranded amplification of the two halves ofthe total sequence due to an excess of the two flanking primers. Insubsequent PCR cycles, these dual asymmetrically amplified fragments,which overlap each other, yield a double-stranded, full-length product.

TBIO synthesis requires only sense-strand primers for the amino-terminalhalf and only antisense-strand primers for the carboxy-terminal half ofa gene sequence. In addition, the TBIO primers may contain identicalregions of temperature-optimized primer overlaps. The TBIO methodinvolves complementation between the next pair of outside primers withthe termini of a fully synthesized inside fragment. TBIO bidirectionalelongation is completed for a given outside primer pair before the nextround of bidirectional elongation takes place.

Successive PCR is a single step PCR approach in which half the senseprimers correspond to one half of the template to be assembled, and theantisense primers correspond to the second half of the template to beassembled. With this approach, bidirectional amplification with an outerprimer pair will not occur until amplification using an inner primerpair is complete.

PDTS typically involves two steps. First individual fragments of the DNAof interest are synthesized: In some embodiments of the invention, 10-12oligonucleotides, such as oligonucleotides of length of about 60, 80,100, 125, 150, 175, 200, 250, 300, 350, or more nucleotides, with about20 bp overlap are mixed and a PCR reaction is carried out with apolymerase, such as pfu DNA, to produce longer DNA fragments. Andsecond, the entire sequence of the DNA of interest is synthesized: 5-10PCR products from the first step are combined and used as the templatefor a second PCR reaction with a polymerase, such as pyrobest DNApolymerase with two outermost oligonucleotides as primers.

Although PCR assembly using short oligonucleotides work well forrelatively shorter nucleic acids, there may be a limit to the maximumnumber of oligonucleotides that can be assembled within a singlereaction. This may impose a size limit on the double stranded DNAproduct. A solution to this problem is to make the DNA in series. Inthis scheme, multiple smaller DNA segments are synthesized in parallelin separate chambers, in multiple chips, or in series and thenintroduced together as precursors for the PCA reaction for assembly intoa “larger” DNA fragment for subsequent PCR amplification. In otherwords, PCR assembly using oligonucleotides would result in a template (afirst-run template) for PCR amplification. A number of first-runtemplates so produced may serve as precursors for PCA assembly of DNAfragments larger than the first-run templates, thus producing second-runtemplates. In turn, the second-run templates may serve as the precursorsfor the assembly of a third-run template, and so on. The approach may berepeated until the desired DNA is obtained.

The oligonucleotides used in the synthesis reactions are typicallysingle stranded molecules for assembling nucleic acids that are longerthan the oligonucleotide itself. An oligonucleotide may be e.g. 20-200,50-300, 75-350 or 100-400 nucleotide building blocks. Those of skill inthe art appreciate that the sequence segment length may fall within anyrange bounded by any of these values (e.g., 20-350 or 200-350). A PCAchamber containing a plurality of oligonucleotides refers to the pool ofoligonucleotides necessary to produce a template corresponding to a geneor to a DNA fragment. When the synthesis reactions and devices are usedin series, the PCA chamber in the subsequent series of reactions wouldcontain a pool of DNA fragments instead of the starting oligonucleotidesfor assembly into templates for PCR. FIG. 12 demonstrates the polymerasecycling assembly of longer constructs from a pool of overlappingoligonucleotides into gradually longer constructs through multiplecycles of the reaction.

It is understood that longer oligonucleotides as described herein can beused advantageously in a variety of gene assembly methods to avoidassembly errors and increase the quality of synthesized genes (FIG. 13).Homologous repeats or high GC regions in a sequence to be assembled mayintroduce errors associated with the correct order and hybridization ofsmaller oligonucleotides. Longer oligonucleotides can circumvent theseproblems by reducing the number of oligonucleotides to be ordered andaligned, by avoiding problematic sequences, such as homology repeats orhigh GC regions from sites of alignment, and/or by reducing the numberof assembly cycles required to assemble the desired gene.

Larger genes may be synthesized combining gene assembly methodshierarchically as exemplified in FIG. 14. Accordingly, a number of genesof intermediary length, for example about 2 kb, can be assembled using afirst gene assembly method, such as PCA. A second gene assembly method,e.g. Gibson Assembly (Gibson et al, Science, 2008, 319, 1215) may beutilized to combine the genes of intermediary length into larger genes,e.g. about 5 or 10 kb. Hierarchical assembly can be applied in stages.In vitro recombination techniques may be used to assemble cassettes ofgene of intermediary length into increasingly longer sequences, e.g.more than 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150,175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 kb or longer.

Oligonucleotides useful for the assembly of genes de novo may besynthesized on one or more solid supports. Exemplary solid supportsinclude, for example, slides, beads, chips, particles, strands, gels,sheets, tubing, spheres, containers, capillaries, pads, slices, films,plates, polymers, or a microfluidic device. Further, the solid supportsmay be biological, nonbiological, organic, inorganic, or combinationsthereof. On supports that are substantially planar, the support may bephysically separated into regions, for example, with trenches, grooves,wells, or chemical barriers (e.g., hydrophobic coatings, etc.). Supportsmay also comprise physically separated regions built into a surface,optionally spanning the entire width of the surface. Suitable supportsfor improved oligonucleotide synthesis are further described herein.

In one aspect, the oligonucleotides may be provided on a solid supportfor use in a microfluidic device, for example, as part of the PCAreaction chamber. Alternatively, oligonucleotides may be synthesized andsubsequently introduced into a microfluidic device.

Generally, the complete gene sequence is broken down into variable orfixed length (N) oligonucleotides as appropriate. A suitableoligonucleotide length can be chosen, e.g. 20-200, 50-300, 75-350 or100-400 nucleotide building blocks. Those of skill in the art appreciatethat the sequence segment length may fall within any range bounded byany of these values (e.g., 20-350 or 200-350). The length of the overlapbetween sub-sequences is about or less than about N/2, but may be chosenas the needs of the assembly reaction dictates, e.g. 6-40 bp, 10-20 bpand 20-30 bp of overlap. Those of skill in the art appreciate that thesequence segment length may fall within any range bounded by any ofthese values (e.g., 20-40 or 6-30). The amount of partial basecomplementarity may vary depending on the assembly method used. Forvarious overlapping gene assembly methods, the PCA oligonucleotides mayoverlap at both the 5′ and 3′ ends, except those forming the ends of theresulting PCR template. Base pair mismatches between oligonucleotidesmay affect hybridization depending on the nature of the mismatch.Mismatches at or near the 3′ end of the oligonucleotide may inhibitextension. However, a G/C rich region of overlap may overcome mismatchesthus resulting in templates containing errors. Accordingly,consideration of the overlap sequence, melting temperature, potentialfor cross-hybridization and secondary structure in oligonucleotidedesign can be taken into consideration.

Nucleic acid sequences resulting from a PCR assembly reaction may bereferred as templates and serve as the target nucleic acid for thereproduction of a complementary strand by PCR. Typically, following anassembly reaction, the PCR assembly products may be double stranded DNAof variable sizes due perhaps to incomplete assembly and/or concatamers.In some embodiments, a first-run template is assembled fromoligo-nucleotides. In other embodiments, a second-run template isassembled from DNA fragments comprising at least two first-runtemplates, the two templates being the PCR reaction products, optionallypurified and/or error-filtered, obtained from the first two runs. Athird-run template is assembled from DNA fragments comprising at leasttwo second-run templates, which may be similarly error-filtered and soon.

Non-polymerase-cycling-assembly-based strategies, such as annealing andligation reaction (Climie and Santi, 1990; Smith et al., 1990; Kalman etal., 1990), insertion gene synthesis (IGS) (Ciccarelli et al., 1990),gene synthesis via one strand (Chen et al., 1990), template-directedligation (TDL) (Strizhov et al., 1996), ligase chain reaction (Au etal., 1998), or any suitable assembly method known in the art may also beused for chemical synthesis of polynucleotides. Othernon-polymerase-cycling-assembly-based gene synthesis strategies include,but are not limited to microarray-based gene synthesis technology (Zhouet al., 2004), Blue Heron solid support technology, Sloning buildingblock technology (Ball, 2004; Schmidt, 2006; Bugl et al., 2007), andRNA-mediated gene assembly from DNA arrays (Wu et al., 2012).

Enzymatic Gene Synthesis

Enzymes that repair single-stranded breaks in double-stranded DNA, firstdiscovered in the 1960s in E. coli and in T4 bacteriophage infected E.coli cells (Meselson, 1964; Weiss and Richardson, 1967; Zimmerman etal., 1967), can be used to join chemically synthesized oligonucleotides,such as deoxyribopolynucleotides, to form continuous bihelicalstructures (Gupta et al., 1968a). In another example, DNA polymerase I(Klenow) can be used to join oligonucleotides to longer polynucleotides.Oligonucleotides can further be joined together via ligation, forexample using a ligase, such as using phage T4 polynucleotide ligase. Insome cases, oligonucleotides can be ligated hierarchically, forminglonger and longer polynucleotides in each step.

Annealing and Ligation Reaction

Another approach for the facile synthesis of genes comprises assembly ofa polynucleotide from many oligonucleotides through annealing andligation reaction (Climie and Santi, 1990; Smith et al., 1990; Kalman etal., 1990). In the first, both strands of the desired sequences can bedivided with short cohesive ends so that adjacent pairs of complementaryoligonucleotides can anneal. The synthesized oligonucleotides can bephosphorylated, for example using a kinase, and annealed before ligationinto a duplex.

Shotgun Ligation and Co-Ligation

The shotgun ligation approach comprises the assembly of a full gene fromseveral synthesized blocks (Eren and Swenson, 1989). Accordingly, a genemay be sub-assembled in several sections, each constructed by theenzymatic ligation of several complementary pairs of chemicallysynthesized oligonucleotides with short single strands complementary tothat of an adjacent pair. Co-ligation of the sections can achieve thesynthesis of the final polynucleotide.

Insertion Gene Synthesis

Insertion gene synthesis (IGS) (Ciccarelli et al., 1990) can be used toassemble a DNA sequence in a stepwise manner within a plasmid containinga single-stranded DNA phage origin of replication. The IGS method isbased upon consecutive targeted insertions of long DNA oligonucleotideswithin a plasmid by oligonucleotide-directed mutagenesis.

Gene Synthesis Via One Strand

Gene synthesis via one strand refers to a method to synthesize a genevia one stand (Chen et al.; 1990). A plus-stranded DNA of the targetgene can be assembled by a stepwise or single-step T4 DNA ligasereaction with several, for example six, oligonucleotides in the presenceof multiple, for example two, terminal complementary oligonucleotidesand multiple, for example three, short interfragment complementaryoligonucleotides. The use of fewer synthesized bases, in comparison tothe double-strand or overlap methods can reduce costs.

Template-Directed Ligation

Template-directed ligation refers to a method to construct largesynthetic genes by ligation of oligonucleotide modules, by partialannealing with a single-stranded DNA template derived from a wild-typegene (Strizhov et al.; 1996). Oligonucleotides comprising only onestrand can be synthesized, in contrast to other technologies thatrequire synthesis of two strands. A ligase, such as the Pfu DNA ligase,can be used to perform thermal cycling for assembly, selection andligation of full-length oligonucleotides as well as for linearamplification of the template-directed ligation (TDL) product. Due toits reliance on a homologous template, this method is suitable to thesynthesis of only a limited number of sequences with similarity to anexisting polynucleotide molecule.

Ligase Chain Reaction

A ligase chain reaction (LCR) can be used method for synthesis ofpolynucleotides (Au et al.; 1998). Fragments can be assembled fromseveral oligonucleotides via ligation, using a ligase, for example PfuDNA ligase. After LCR, the full-length gene can be amplified with themixture of fragments which shared an overlap by denaturation andextension using the outer two oligonucleotides.

Microarray-Mediated Gene Synthesis

Microarray-mediated gene synthesis, as a general concept, is based onthe capacity to immobilize tens of thousands of specific probes on asmall solid surface (Lockhart and Barlow, 2001). For the production ofarrays, DNA can either be synthesized directly on the solid support(Lipshutz et al., 1999; Hughes et al., 2001) or can be deposited in apre-synthesized form onto the surface, for example with pins or ink-jetprinters (Goldmann and Gonzalez, 2000). The oligonucleotides obtainedcan be used in ligation under thermal cycling conditions to generate DNAconstructs of several hundreds of base-pairs. Another microchip-basedtechnology for accurate multiplex gene synthesis, the modifiedarray-mediated gene synthesis technology (Tian et al., 2004), is similarto amplification and assembly of chip-eluted DNA AACED), a methoddeveloped for high-throughput gene synthesis (Richmond et al., 2004).Pools of thousands of ‘construction’ oligonucleotides and taggedcomplementary ‘selection’ oligonucleotides can be synthesized onphoto-programmable microfluidic chips, released, ligation amplified, andselected by hybridization to reduce synthesis errors (Tian et al.,2004).

Blue Heron Technology

The Blue Heron technology, developed by Blue Heron Biotechnology, isbased on a solid-phase support strategy based on the GeneMaker platformand enables automation (Parker and Mulligan, 2003; Mulligan and Tabone,2003; Mulligan et al., 2007). The GeneMaker protocol may generallycomprise a user sequence data entry, an algorithm designing suitableoligonucleotides for the assembly of entered sequence, oligonucleotidessynthesis and hybridization into duplexes, automated ligation basedsolid-phase assembly through automated sequential additions inside acolumn on a solid support matrix, and/or cloning and sequenceverification. The Blue Heron technology relies on the sequentialaddition of building blocks to lower errors that occur with other geneassembly methods based on non-serial pools of building blocks, such asPCR methods.

Various embodiments of the invention make use of serial and hierarchicalassembly methods as exemplified in the implementation of the Blue Herontechnology.

Sloning Building Block Technology

Sloning building block technology (Slonomics™; Sloning BiotechnologyGmbH, Puchheim, Germany) is another method using a ligation-basedstrategy for chemical gene synthesis (Adis International, 2006). TheSloning synthesis method consists of a series of parallel iterative andstandardized reaction steps (pipetting, mixing, incubation, washing)(Schatz and O'Connell, 2003; Schatz et al., 2004; Schatz, 2006). Incontrast to ligating oligonucleotides specifically designed andsynthesized for a given gene construct, Sloning technology uses alibrary of standardized building blocks that can be combined to form anydesired sequence with a series of standardized, fully automated,cost-effective reaction steps (Schatz and O'Connell, 2003; Schatz,2006).

Golden Gate Assembly

The Golden-gate method (see, e.g., Engler et al. (2008) PLoS ONE, 3(11):e3647; Engler et al. (2009) PLoS ONE 4(5): e5553) offers standardized,multi-part DNA assembly. The Golden-gate method can use Type IIsendonucleases, whose recognition sites are distal from their cuttingsites. There are several different Type IIs endonucleases to choosefrom, for example BsaI. The Golden-gate method can be advantageous bythe use of a single Type IIs endonuclease. The Golden-gate method isfurther described in U.S. Patent Pub. 2012/0258487, which isincorporated herein by reference in its entirety.

In some cases, the methods and compositions for gene assembly mayinvolve a combination of specifically synthesized building blocks andpresynthesized building blocks. Libraries of presynthesizedoligonucleotides may be stored and assembly processes for desired targetnucleic acids may be optimized for maximum use of presynthesizedoligonucleotides, minimizing the need for new synthesis. Specificallysynthesized oligonucleotides may fill in parts of a target nucleic acid,for which there is no coverage in libraries of presynthesizedoligonucleotides.

RNA-Mediated Gene Assembly

In various embodiments, RNA-mediated gene assembly is used to assembleRNA transcripts from DNA elements, optionally immobilized to a surfaceforming an immobilized DNA array. DNA elements are designed to includean RNA polymerase (RNAP) promoter sequence, such as a T& RNA polymerasepromoter sequence, toward the 5′ end. Hybridization of anoligonucleotide encoding the promoter sequence, such as the T7 RNAPpromoter sequence, to a DNA element can yield a double-strandedpromoter. Addition of RNAP may affect the transcription from theseoptionally surface-bound promoters yielding many RNA copies. Theseamplified RNA molecules can be designed to allow self-assembly to yielda longer RNA. Briefly, the DNA elements can be designed to encode“segment sequences”, which are the sections of the desired full-lengthRNA transcript, and “splint sequences”, which are complementary RNAsthat serve as templates to direct the correct assembly of the RNAsegments. The DNA elements encoding RNA segments or splints may bechosen to optimize one or more reactions during the synthesis ofassembled polynucleotides. For example, the DNA elements may beconstructed such that that the 5′ end of each RNA transcript correspondsto a GG dinucleotide, which is believed to affect higher efficiency oftranscription exhibited by T7 RNA polymerase (T7 RNAP). GGGtrinucleotide sequences at the 5′ terminus may in turn be avoided, toavoid giving rise to a ladder of poly G transcripts in which the numberof G residues can range from 1-3, attributed to “slippage” of the enzymeduring coupling of GTP. Assembly can be affected via RNA:RNAhybridization of the segments to the splints. Nicks can be sealedchemically or enzymatically, using a suitable enzyme known in the art.In one example, the assembly of the RNA segment sequences into thefull-length RNA transcript includes ligation with T4 RNA ligase 2.Triphosphorylated transcripts, such as those generated by T7 RNApolymerase can be “trimmed” to their monophosphorylated analogues beforeligation. Trimming can be accomplished by treatment of the transcriptpool with RNA 5′ pyrophosphohydrolase removing a pyrophosphate groupfrom the 5′ end of each RNA. The transcript, once synthesized, can becopied by reverse transcription polymerase chain reaction (RT-PCR) toyield the corresponding gene. The assembled RNA sequence or its DNAequivalent may be amplified using a suitable nucleic acid amplificationmethod, including those described elsewhere herein. The method isfurther described in Wu et al. (Cheng-Hsien Wu, Matthew R. Lockett, andLloyd M. Smith, RNA-Mediated Gene Assembly from DNA Arrays, 2012, Angew.Chem. Int. Ed. 51, 4628-4632), which is herein incorporated by referencein its entirety.

Nonenzymatic Chemical Ligation of DNA

Other approaches include, nonenzymatic chemical ligation of DNA, forexample with cyanogen bromide as a condensing agent, as described forthe synthesis of a 183 bp biologically active mini-gene (Shabarova etal., 1991).

In some embodiments, assembly of oligonucleotides comprises the use ofCLICK chemistry. Suitable methods to link various molecules using CLICKchemistry are known in the art (for CLICK chemistry linkage ofoligonucleotides, see, e.g. El-Sagheer et al. (PNAS, 108:28,11338-11343, 2011). Click chemistry may be performed in the presence ofCu1.

Error Rates and Corrections

A critical limitation of current gene synthesis technology is the lowsequence fidelity of the process: gene clones created from chemicallysynthesized DNA often contain sequence errors. These errors can beintroduced at many stages of the process: during chemical synthesis ofthe component oligonucleotides, during assembly of the double-strandedoligonucleotides, and by chemical damage occurring during themanipulation and isolation of the DNA or during the cloning process.

Known methods generating chemically-synthesized DNA fragments have veryhigh sequence error rates, e.g. every 200 to 500 bp on average. Themethods described herein allow for the initial de novo synthesis ofoligonucleotides and longer polynucleotide with very low error rates.Common mutations in oligonucleotides comprise deletions that can comefrom capping, oxidation and/or deblocking failure. Other prominent sidereactions include modification of guanosine (G) by ammonia to give2,6-diaminopurine, which codes as an adenosine (A). Deamination is alsopossible with cytidine (C) forming uridine (U) and adenosine forminginosine (I).

Without being bound by theory, non limiting examples of basemodifications typically produced during the synthesis of anoligonucleotide using the phosphoramidite method include transaminationof the 06-oxygen of deoxyguanosine to form a 2,6-diaminopurine residue,deamination of the N4-amine of deoxycytidine to form a uridine residue(Eadie, J. S. and Davidson, D. S., Nucleic Acids Res. 15:8333, 1987),depurination of N6-benzoyldeoxyadenosine yielding an apurinic site(Shaller, H. and Khorana, H. G., J. Am. Chem. Soc. 85:3828, 1963;Matteucci, M. D. and Caruthers, M. H., J. Am. Chem. Soc. 103:3185,1981), and incomplete removal of the N2-isobutyrlamide protecting groupon deoxyguanosine. Each of these side products (byproducts) cancontribute to sequence errors in cloned synthetic polynucleotides.

In addition, common methods of oligonucleotide synthesis are prone tothe formation of truncated products that are less than the full lengthof the desired oligonucleotide. The solid phase approach tooligonucleotide synthesis involves building an oligomer chain that isanchored to a solid support typically through its 3′-hydroxyl group, andis elongated by coupling of building blocks to its 5′ end. The yield ofeach coupling step in a given chain-elongation cycle will generally be<100%. For an oligonucleotide of length n, there are n−1 linkages andthe maximum yield estimation will typically be governed by [couplingefficiency]^(n-1). For a 25-mer, assuming a coupling efficiency of 98%,the calculated maximum yield of full-length product will be around 61%.The final product therefore would contain decreasing amounts of n−1,n−2, n−3 etc. failure sequences.

Another class of synthetic failures is the formation of “n+” productsthat are longer than the full length of the desired oligonucleotide.Without being bound by theory, these products may originate from thebranching of the growing oligonucleotide, in which a phosphoramiditemonomer reacts through the bases, especially the N-6 of adenosine andthe O-6 of guanosine. Another source of n+ products is the initiationand propagation from unwanted reactive sites on the solid support. Then+ products may also form if the 5′-trityl protecting group isinadvertently deprotected during the coupling step. This prematureexposure of the 5′-hydroxyl allows for a double addition of aphosphoramidite. This type of synthetic failure of the oligonucleotidesynthesis process can also contribute to sequence errors in syntheticgenes. Methods and compositions of the invention, in variousembodiments, allow for reducing errors during de novo synthesis ofoligonucleotides through precise control of reaction parameters asdescribed in further detail elsewhere herein.

Other types of errors maybe introduced during the assembly ofoligonucleotides into longer constructs during PCR-based as well asnon-PCR-based assembly methods. For example, ligation of syntheticdouble-stranded oligonucleotides to other synthetic double-strandedoligonucleotides to form larger synthetic double-strandedoligonucleotides may be prone to errors. For example, T4 DNA ligaseexhibits poor fidelity, sealing nicks with 3′ and 5′ A/A or T/Tmismatches (Wu, D. Y., and Wallace, R. B., Gene 76:245-54, 1989), 5′ G/Tmismatches (Harada, K. and Orgel, L. Nucleic Acids Res. 21:2287-91,1993) or 3′ C/A, C/T, T/G, T/T, T/C, A/C, G/G or G/T mismatches(Landegren, U., Kaiser, R., Sanders, J., and Hood, L., Science241:1077-80, 1988).

The error rate also limits the value of gene synthesis for theproduction of libraries of gene variants. With an error rate of 1/300,about 0.7% of the clones in a 1500 base pair gene will be correct. Asmost of the errors from oligonucleotide synthesis result in frame-shiftmutations, over 99% of the clones in such a library will not produce afull-length protein. Reducing the error rate by 75% would increase thefraction of clones that are correct by a factor of 40. The methods andcompositions of the invention allow for fast de novo synthesis of largeoligonucleotide and gene libraries with error rates that are lower thancommonly observed gene synthesis methods both due to the improvedquality of synthesis and the applicability of error correction methodsthat are enabled in a massively parallel and time-efficient manner.Accordingly, libraries may be synthesized with base insertion, deletion,substitution, or total error rates that are under 1/300, 1/400, 1/500,1/600, 1/700, 1/800, 1/900, 1/1000, 1/1250, 1/1500, 1/2000, 1/2500,1/3000, 1/4000, 1/5000, 1/6000, 1/7000, 1/8000, 1/9000, 1/10000,1/12000, 1/15000, 1/20000, 1/25000, 1/30000, 1/40000, 1/50000, 1/60000,1/70000, 1/80000, 1/90000, 1/100000, 1/125000, 1/150000, 1/200000,1/300000, 1/400000, 1/500000, 1/600000, 1/700000, 1/800000, 1/900000,1/1000000, or less, across the library, or across more than 80%, 85%,90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.98%,99.99%, or more of the library. The methods and compositions of theinvention further relate to large synthetic oligonucleotide and genelibraries with low error rates associated with at least 30%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, 99.9%, 99.95%, 99.98%, 99.99%, or more of the oligonucleotides orgenes in at least a subset of the library to relate to error freesequences in comparison to a predetermined/preselected sequence. In someembodiments, at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%, 99.98%, 99.99%, ormore of the oligonucleotides or genes in an isolated volume within thelibrary have the same sequence. In some embodiments, at least 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, 99.9%, 99.95%, 99.98%, 99.99%, or more of any oligonucleotides orgenes related with more than 95%, 96%, 97%. 98%, 99%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or more similarity or identity have the samesequence. In some embodiments, the error rate related to a specifiedlocus on an oligonucleotide or gene is optimized. Thus, a given locus ora plurality of selected loci of one or more oligonucleotides or genes aspart of a large library may each have an error rate that is less than1/300, 1/400, 1/500, 1/600, 1/700, 1/800, 1/900, 1/1000, 1/1250, 1/1500,1/2000, 1/2500, 1/3000, 1/4000, 1/5000, 1/6000, 1/7000, 1/8000, 1/9000,1/10000, 1/12000, 1/15000, 1/20000, 1/25000, 1/30000, 1/40000, 1/50000,1/60000, 1/70000, 1/80000, 1/90000, 1/100000, 1/125000, 1/150000,1/200000, 1/300000, 1/400000, 1/500000, 1/600000, 1/700000, 1/800000,1/900000, 1/1000000, or less. In various embodiments, such erroroptimized loci may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 100000, 500000,1000000, 2000000, 3000000 or more loci. The error optimized loci may bedistributed to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10000, 100000, 500000, 1000000,2000000, 3000000 or more oligonucleotides or genes.

The error rates can be achieved with or without error correction. Theerror rates can be achieved across the library, or across more than 80%,85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 99.95%,99.98%, 99.99%, or more of the library.

The library may comprise more than 100, 200, 300, 400, 500, 600, 750,1000, 15000, 20000, 30000, 40000, 50000, 60000, 75000, 100000, 200000,300000, 400000, 500000, 600000, 750000, 1000000, 2000000, 3000000,4000000, 5000000, or more different oligonucleotides or genes. Thedifferent oligonucleotides or genes may be related topredetermined/preselected sequences. The library may compriseoligonucleotides or genes that are over 500 bp, 600 bp, 700 bp, 800 bp,900 bp, 1000 bp, 1250 bp, 1500 bp, 1750 bp, 2000 bp, 2500 bp, 3000 bp,4000 bp, 5000 bp, 6000 bp, 7000 bp, 8000 bp, 9000 bp, 10 kb, 20 kb, 30kb, 40 kb, 50 kb, 60 kb, 80 kb, 90 kb, 100 kb long, or longer. It isunderstood that the library may comprise of a plurality of differentsubsections, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 subsections or more,that are governed by different error rates and/or construct sizes.Compositions and methods of the invention further allow construction ofthe above mentioned large synthetic libraries of oligonucleotides orgenes with low error rates described above in short time frames, such usin less than three months, two months, one month, three weeks, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or less. Genes of the abovementioned libraries maybe synthesized by assembling de novo synthesizedolignucleotides by suitable gene assembly methods further described indetail elsewhere herein or otherwise known in the art.

Several methods are known in the art for removal of error-containingsequences in a synthesized gene. A DNA mismatch-binding protein, MutS(from Thermus aquaticus), can be employed to remove failure productsfrom synthetic genes using different strategies (Schofield and Hsieh,2003; Carr et al., 2004; Binkowski et al., 2005). Some other strategies(Pogulis et al., 1996; Ling and Robinson, 1997; An et al., 2005; Peng etal., 2006b) use site-directed mutagenesis by overlap extension PCR tocorrect mistakes, and can be coupled with two or more rounds of cloningand sequencing, as well as additional synthesis of oligonucleotides.Functional selection and identification after gene synthesis is anotherapproach (Xiong et al., 2004b; Smith et al., 2003). Another approach toerror correction uses SURVEYOR endonuclease (Transgenomic), amismatch-specific DNA endonuclease to scan for known and unknownmutations and polymorphisms in heteroduplex DNA. SURVEYOR technology isbased on a mismatch-specific DNA endonuclease from celery, Surveyornuclease, which is a member of the CEL nuclease family of plant DNAendonucleases (Qiu et al., 2004). Surveyor nuclease cleaves with highspecificity at the 3′ side of any base-substitution mismatch and otherdistortion site in both DNA strands, including all base substitutionsand insertion/deletions up to at least 12 nucleotides.Insertion/deletion mismatches and all base-substitution mismatches canbe recognized, with varying efficiency of cleavage based on the mismatchsequence. In one example, Surveyor nuclease technology can be used formismatch detection in a method involving four steps: (i) optionalpolynucleotide amplification, e.g. PCR, of desired polynucleotidetargets with both mutant/variant and wild-type/desired sequences; (ii)hybridization resulting heteroduplexes comprising mismatches; (iii)treatment of heteroduplexes with Surveyor nuclease to cleave at mismatchsites; and (iv) optional analysis of digested polynucleotide productsusing the detection/separation platform of choice (FIGS. 15-16). Thecleavage products resulting from the treatment of heteroduplexes may besubjected to PCA after the error at the cleavage site is chewed out,e.g. by an exonuclease, to generate error depleted products (FIG. 15).The mismatch bases can be substantially or in some cases completelyremoved to produce error-free strands. In some embodiments, the cleavedstrands can be reannealed to targets in a pool of polynucleotides andextended. As the frequency of error containing polynucleotides is verylow after the initial annealing and cleavage of heteroduplexes removingmismatches, most cleaved strands will anneal to targets with sequencesfree of error at the site of the initial mismatch. Through extensionalong the targets, polynucleotides can be resynthesized free of theinitial mismatch. Various examples of gene assembly incorporate errorcorrection. For example, the PCR-based accurate synthesis (PAS) protocolcan incorporate: design of the gene and oligonucleotides, purificationof the oligonucleotides, a first PCR to synthesize segments, a secondPCR to assemble the full-length gene, and sequencing and errorcorrection (Xiong et al., 2006). Alternatively, the sample by besubjected to PCR, wherein the cleaved products are not able toparticipate, thereby diluting the abundance of the error in the sample(FIG. 16).

In certain embodiments, the present invention provides methods thatselectively remove double-stranded oligonucleotides, such as DNAmolecules, with mismatches, bulges and small loops, chemically alteredbases and other heteroduplexes arising during the process of chemicalsynthesis of DNA, from solutions containing perfectly matched syntheticDNA fragments. The methods separate specific protein-DNA complexesformed directly on heteroduplex DNA or through an affinity systemcomprising an incorporated nucleotide analog, e.g. one that is based onavidin-biotin-DNA complexes formed following the introduction of abiotin molecule or a biotin analog, into heteroduplex containing DNA andsubsequent binding by any member of the avidin family of proteins,including streptavidin. The avidin may be immobilized on a solidsupport.

Central to the method are enzymes that recognize and bind specificallyto mismatched, or unpaired bases within a double-strandedoligonucleotide (e.g., DNA) molecule and remain associated at or near tothe site of the heteroduplex, create a single or double strand break orare able to initiate a strand transfer transposition event at or near tothe heteroduplex site. The removal of mismatched, mispaired andchemically altered heteroduplex DNA molecules from a synthetic solutionof DNA molecules results in a reduced concentration of DNA moleculesthat differ from the expected synthesized DNA sequence.

The mismatch recognition proteins typically bind on or within thevicinity of a mismatch. Reagents for mismatch recognition protein basederror correction may comprise proteins that are endonucleases,restriction enzymes, ribonucleases, mismatch repair enzymes, resolvases,helicases, ligases, antibodies specific for mismatches, and theirvariants. The enzymes can be selected, for example, from T4 endonuclease7, T7 endonuclease 1, S1, mung bean endonuclease, MutY, MutS, MutH,MutL, cleavase, and HINF1. In certain embodiments of the invention, amismatch recognition protein cleaves at least one strand of themismatched DNA in the vicinity of the mismatch site.

In the case of proteins that recognize and cleave heteroduplex DNAforming a single strand nick, for example the CELI endonuclease enzyme,the resultant nick can be used as substrate for DNA polymerase toincorporate modified nucleotides suitable for affinity partnerships,e.g. ones containing a biotin moiety or an analog thereof. There aremany examples of proteins that recognize mismatched DNA and produce asingle strand nick, including resolvase endonucleases, glycosylases andspecialized MutS-like proteins that possess endonuclease activity. Insome cases the nick is created in a heteroduplex DNA molecule afterfurther processing, for example thymine DNA glycosylases can be used torecognize mismatched DNA and hydrolyze the bond between deoxyribose andone of the bases in DNA, generating an abasic site without necessarilycleaving the sugar phosphate backbone of DNA. The abasic site can beconverted by an AP endonuclease to a nicked substrate suitable for DNApolymerase extension. Protein-heteroduplex DNA complexes can thus beformed directly, in the example of MutS proteins, or indirectly,following incorporation of nucleotide analogs, e.g. biotin or analogsthereof, into the heteroduplex containing strand and subsequent bindingof biotin or biotin analogs with streptavidin or avidin proteins.

Other error correction methods may rely on transposase enzymes, such asthe MuA transposase, preferentially inserting labeled DNA, e.g. biotinor biotin-analog labeled DNA, containing a precleaved version of thetransposase DNA binding site into or near to the site of mismatched DNAin vitro via a strand transfer reaction. The in vitro MuA transposasedirected strand transfer is known by those skilled in the art andfamiliar with transposase activity to be specific for mismatched DNA. Inthis method, the precleaved MuA binding site DNA may be biotinylated atthe 5′ end of the molecule enabling the formation of aprotein-biotin-DNA complex with streptavidin or avidin protein followingstrand transfer into heteroduplex containing DNA.

Separation of protein-DNA complexes in vitro can be achieved byincubation of the solution containing protein-DNA complexes with a solidmatrix that possesses high affinity and capacity for binding of proteinand low affinity for binding of DNA. In some cases, such matrices can beembedded within microfluidic devices in connection with the variousembodiments of the invention described herein.

Several large classes of enzymes preferentially digest heteroduplexpolynucleotides, such as DNA substrates, containing mismatches,deletions or damaged bases. Typically, these enzymes act to converttheir damaged or mismatched substrates into nicks or single base pairgaps (in some cases with the help of an AP endonuclease that convertsabasic sites into nicks). DNA glycosylases, mismatch endonucleases, andthe MutSLH mismatch repair proteins are especially useful for theirutility in modifying synthetic fragments which contain errors. Methodsand compositions of the present invention may rely on these nicks orsmall gaps to identify the error-containing DNA molecules and removethem from the cloning process.

A combination of techniques can be used for removing the treatedpolynucleotides containing errors. DNA glycosylases are a class ofenzymes that remove mismatched bases and, in some cases, cleave at theresulting apurinic/apyrimidimic (AP) site. Thymine DNA glycosylases(TDGs) can be used to enrich mismatch-containing or perfectly-matchedDNA populations from complex mixtures (X. Pan and S. Weissman, “Anapproach for global scanning of single nucleotide variations” 2002 PNAS99:9346-9351). DNA glycosylases can be used to hydrolyze the bondbetween deoxyribose and one of the bases in DNA, generating an abasicsite without necessarily cleaving the sugar phosphate backbone of DNA.All four groups of single base mismatches and some other mismatchescould be hydrolyzed by a mixture of two TDGs. In addition, the enzymes'high affinity for abasic sites in the absence of magnesium can beutilized to separate DNA molecules into populations enriched or depletedfor heteroduplexes. A very large number of DNA glycosylases have beenidentified, and non-limiting examples can be found in US Pat. Pub.2006/0134638, which is incorporated herein by reference in its entirety.DNA glycosylases typically act on a subset of unnatural, damaged ormismatched bases, removing the base and leaving a substrate forsubsequent repair. As a class, the DNA glycosylases have broad, distinctand overlapping specificities for the chemical substrates that they willremove from DNA. Glycosylase treatment may be especially useful inreducing the error rates of base substitutions to low levels.Glycosylases that leave AP sites are combined with an AP endonucleasesuch as E. coli Endonuclease IV or Exo III to generate a nick in theDNA.

Non-limiting examples of mismatch endonuclease enzymes for nicking DNAin the region of mismatches or damaged DNA include T7 Endonuclease I, E.coli Endonuclease V, T4 Endonuclease VII, mung bean nuclease, Cell, E.coli Endonuclease IV, and UVDE.

The use of the MutSLH complex to remove the majority of errors from PCRfragments is described by Smith et al. (J. Smith and P. Modrich,“Removal of polymerase-produced mutant sequences from PCR products.”1997, PNAS 94:6847-6850), incorporated herein by reference in itsentirety. In the absence of DAM methylation, the MutSLH complex can beused to catalyze double-stranded cleavage at (GATC) sites. PCR productscan be treated with MutSLH in the presence of ATP.

A more detailed disclosure regarding error correction in syntheticpolynucleotides can be found in U.S. Pat. Pub. 2006/0134638 and U.S.Pat. No. 6,664,112, both of which are herein incorporated in theirentirety.

Enzymes, binding partners and other reagents used in error correction ofsynthesized polynucleotides according to the methods and compositions ofthe invention may be immobilized on surfaces, such as coated orfunctionalized surfaces, on supports and substrates described herein.Reactions can be carried out in situ with one or more componentsimmobilized. Purification schemes enriching polynucleotides with feweror no errors utilizing such components on appropriate surfaces areunderstood to be within the bounds of the invention.

Ultimately, strategies for gene assembly rely on high-qualityoligonucleotides to achieve the de novo synthesis of polynucleotideswith low error rates. Methods and compositions described herein allowfor the synthesis of such high-quality oligonucleotides in variousembodiments.

Amplification of Nucleic Acids

In some embodiments, the nucleic acids described herein are amplified.Amplification can be performed by any means known in the art. In somecases, the nucleic acids are amplified by polymerase chain reaction(PCR). Various PCR methods are known in the art, as described in, forexample, U.S. Pat. Nos. 5,928,907 and 6,015,674, the completedisclosures of which are hereby incorporated by reference for anypurpose. Other methods of nucleic acid amplification include, forexample, ligase chain reaction, oligonucleotide ligations assay, andhybridization assay. These and other methods are described in greaterdetail in U.S. Pat. Nos. 5,928,907 and 6,015,674. Real-time opticaldetection systems are known in the art, as also described in greaterdetail in, for example, U.S. Pat. Nos. 5,928,907 and 6,015,674,incorporated herein above. Other amplification methods that can be usedherein include those described in U.S. Pat. Nos. 5,242,794; 5,494,810;4,988,617; and 6,582,938, all of which are incorporated herein in theirentirety.

In some aspects of the invention, exponential amplification of nucleicacids or polynucleotides is used. These methods often depend on theproduct catalyzed formation of multiple copies of a nucleic acid orpolynucleotide molecule or its complement. The amplification productsare sometimes referred to as “amplicons.” One such method for theenzymatic amplification of specific double stranded sequences of DNA ispolymerase chain reaction (PCR). This in vitro amplification procedureis based on repeated cycles of denaturation, oligonucleotide primerannealing, and primer extension by thermophilic template dependentpolynucleotide polymerase, resulting in the exponential increase incopies of the desired sequence of the polynucleotide analyte flanked bythe primers. The two different PCR primers, which anneal to oppositestrands of the DNA, are positioned so that the polymerase catalyzedextension product of one primer can serve as a template strand for theother, leading to the accumulation of a discrete double strandedfragment whose length is defined by the distance between the 5′ ends ofthe oligonucleotide primers. Other amplification techniques that can beused in the methods of the provided invention include, e.g., AFLP(amplified fragment length polymorphism) PCR (see e.g.: Vos et al. 1995.AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23:4407-14), allele-specific PCR (see e.g., Saiki R K, Bugawan T L, Horn GT, Mullis K B, Erlich H A (1986). Analysis of enzymatically amplifiedbeta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotideprobes Nature 324: 163-166), Alu PCR, assembly PCR (see e.g., Stemmer WP, Crameri A, Ha K D, Brennan T M, Heyneker H L (1995). Single-stepassembly of a gene and entire plasmid from large numbers ofoligodeoxyribonucleotides Gene 164: 49-53), assymetric PCR (see e.g.,Saiki R K supra), colony PCR, helicase dependent PCR (see e.g., MyriamVincent, Yan Xu and Huimin Kong (2004). Helicase-dependent isothermalDNA amplification EMBO reports 5 (8): 795-800), hot start PCR, inversePCR (see e.g., Ochman H, Gerber A S, Hartl D L. Genetics. 1988 November;120(3):621-3), in situ PCR, intersequence-specific PCR or IS SR PCR,digital PCR, linear-after-the-exponential-PCR or Late PCR (see e.g.,Pierce K E and Wangh L T (2007). Linear-after-the-exponential polymerasechain reaction and allied technologies Real-time detection strategiesfor rapid, reliable diagnosis from single cells Methods Mol. Med. 132:65-85), long PCR, nested PCR, real-time PCR, duplex PCR, multiplex PCR,quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplexfluorescent PCR (MF-PCR), restriction fragment length polymorphism PCR(PCR-RFLP), PCK-RFLPIRT-PCR-IRFLP, polonony PCR, in situ rolling circleamplification (RCA), bridge PCR, picotiter PCR and emulsion PCR, orsingle cell PCR. Other suitable amplification methods include,transcription amplification, self-sustained sequence replication,selective amplification of target polynucleotide sequences, consensussequence primed polymerase chain reaction (CP-PCR), arbitrarily primedpolymerase chain reaction (AP-PCR), and degenerateoligonucleotide-primed PCR (DOP-PCR). Another method for amplificationinvolves amplification of a single stranded polynucleotide using asingle oligonucleotide primer. The single stranded polynucleotide thatis to be amplified contains two non-contiguous sequences that aresubstantially or completely complementary to one another and, thus, arecapable of hybridizing together to form a stem-loop structure. Thissingle stranded polynucleotide already may be part of a polynucleotideanalyte or may be created as the result of the presence of apolynucleotide analyte.

Another method for achieving the result of an amplification of nucleicacids is known as the ligase chain reaction (LCR). This method uses aligase enzyme to join pairs of preformed nucleic acid probes. The probeshybridize with each complementary strand of the nucleic acid analyte, ifpresent, and ligase is employed to bind each pair of probes togetherresulting in two templates that can serve in the next cycle to reiteratethe particular nucleic acid sequence.

Another method for achieving nucleic acid amplification is the nucleicacid sequence based amplification (NASBA). This method is apromoter-directed, enzymatic process that induces in vitro continuous,homogeneous and isothermal amplification of a specific nucleic acid toprovide RNA copies of the nucleic acid. The reagents for conductingNASBA include a first DNA primer with a 5′-tail comprising a promoter, asecond DNA primer, reverse transcriptase, RNase-H, T7 RNA polymerase,NTPs and dNTPs.

Another method for amplifying a specific group of nucleic acids is theQ-beta-replicase method, which relies on the ability of Q-beta-replicaseto amplify its RNA substrate exponentially. The reagents for conductingsuch an amplification include “midi-variant RNA” (amplifiablehybridization probe), NTP's, and Q-beta-replicase.

Another method for amplifying nucleic acids is known as 3SR and issimilar to NASBA except that the RNase-H activity is present in thereverse transcriptase. Amplification by 3SR is an RNA specific targetmethod whereby RNA is amplified in an isothermal process combiningpromoter directed RNA polymerase, reverse transcriptase and RNase H withtarget RNA. See for example Fahy et al. PCR Methods Appl. 1:25-33(1991).

Another method for amplifying nucleic acids is the TranscriptionMediated Amplification (TMA) used by Gen-Probe. The method is similar toNASBA in utilizing two enzymes in a self-sustained sequence replication.See U.S. Pat. No. 5,299,491 herein incorporated by reference.

Another method for amplification of nucleic acids is Strand DisplacementAmplification (SDA) (Westin et al 2000, Nature Biotechnology, 18,199-202; Walker et al 1992, Nucleic Acids Research, 20, 7, 1691-1696),which is an isothermal amplification technique based upon the ability ofa restriction endonuclease such as HincII or BsoBI to nick theunmodified strand of a hemiphosphorothioate form of its recognitionsite, and the ability of an exonuclease deficient DNA polymerase such asKlenow exo minus polymerase, or Bst polymerase, to extend the 3′-end atthe nick and displace the downstream DNA strand. Exponentialamplification results from coupling sense and antisense reactions inwhich strands displaced from a sense reaction serve as targets for anantisense reaction and vice versa.

Another method for amplification of nucleic acids is Rolling CircleAmplification (RCA) (Lizardi et al. 1998, Nature Genetics, 19:225-232).RCA can be used to amplify single stranded molecules in the form ofcircles of nucleic acids. In its simplest form, RCA involves thehybridization of a single primer to a circular nucleic acid. Extensionof the primer by a DNA polymerase with strand displacement activityresults in the production of multiple copies of the circular nucleicacid concatenated into a single DNA strand.

In some embodiments of the invention, RCA is coupled with ligation. Forexample, a single oligonucleotide can be used both for ligation and asthe circular template for RCA. This type of polynucleotide can bereferred to as a “padlock probe” or a “RCA probe.” For a padlock probe,both termini of the oligonucleotide contain sequences complementary to adomain within a nucleic acid sequence of interest. The first end of thepadlock probe is substantially complementary to a first domain on thenucleic acid sequence of interest, and the second end of the padlockprobe is substantially complementary to a second domain, adjacent to thefirst domain near the first domain. Hybridization of the oligonucleotideto the target nucleic acid results in the formation of a hybridizationcomplex. Ligation of the ends of the padlock probe results in theformation of a modified hybridization complex containing a circularpolynucleotide. In some cases, prior to ligation, a polymerase can fillin the gap by extending one end of the padlock probe. The circularpolynucleotide thus formed can serve as a template for RCA that, withthe addition of a polymerase, results in the formation of an amplifiedproduct nucleic acid. The methods of the invention described herein canproduce amplified products with defined sequences on both the 5′- and3′-ends. Such amplified products can be used as padlock probes.

Some aspects of the invention utilize the linear amplification ofnucleic acids or polynucleotides. Linear amplification generally refersto a method that involves the formation of one or more copies of thecomplement of only one strand of a nucleic acid or polynucleotidemolecule, usually a nucleic acid or polynucleotide analyte. Thus, theprimary difference between linear amplification and exponentialamplification is that in the latter process, the product serves assubstrate for the formation of more product, whereas in the formerprocess the starting sequence is the substrate for the formation ofproduct but the product of the reaction, i.e. the replication of thestarting template, is not a substrate for generation of products. Inlinear amplification the amount of product formed increases as a linearfunction of time as opposed to exponential amplification where theamount of product formed is an exponential function of time.

In some embodiments, amplification methods can be solid-phaseamplification, polony amplification, colony amplification, emulsion PCR,bead RCA, surface RCA, surface SDA, etc., as will be recognized by oneof skill in the art. In some embodiments, amplification methods thatresults in amplification of free DNA molecules in solution or tetheredto a suitable matrix by only one end of the DNA molecule can be used.Methods that rely on bridge PCR, where both PCR primers are attached toa surface (see, e.g., WO 2000/018957 and Adessi et al., Nucleic AcidsResearch (2000): 28(20): E87) can be used. In some cases the methods ofthe invention can create a “polymerase colony technology,” or “polony.”referring to a multiplex amplification that maintains spatial clusteringof identical amplicons (see Harvard Molecular Technology Group andLipper Center for Computational Genetics website). These include, forexample, in situ polonies (Mitra and Church, Nucleic Acid Research 27,e34, Dec. 15, 1999), in situ rolling circle amplification (RCA) (Lizardiet al., Nature Genetics 19, 225, July 1998), bridge PCR (U.S. Pat. No.5,641,658), picotiter PCR (Leamon et al., Electrophoresis 24, 3769,November 2003), and emulsion PCR (Dressman et al., PNAS 100, 8817, Jul.22, 2003). The methods of the invention provide new methods forgenerating and using polonies.

Amplification may be achieved through any process by which the copynumber of a target sequence is increased, e.g. PCR. Conditions favorableto the amplification of target sequences by PCR are known in the art,can be optimized at a variety of steps in the process, and depend oncharacteristics of elements in the reaction, such as target type, targetconcentration, sequence length to be amplified, sequence of the targetand/or one or more primers, primer length, primer concentration,polymerase used, reaction volume, ratio of one or more elements to oneor more other elements, and others, some or all of which can be altered.In general, PCR involves the steps of denaturation of the target to beamplified (if double stranded), hybridization of one or more primers tothe target, and extension of the primers by a DNA polymerase, with thesteps repeated (or “cycled”) in order to amplify the target sequence.Steps in this process can be optimized for various outcomes, such as toenhance yield, decrease the formation of spurious products, and/orincrease or decrease specificity of primer annealing. Methods ofoptimization are well known in the art and include adjustments to thetype or amount of elements in the amplification reaction and/or to theconditions of a given step in the process, such as temperature at aparticular step, duration of a particular step, and/or number of cycles.In some embodiments, an amplification reaction comprises at least 5, 10,15, 20, 25, 30, 35, 50, or more cycles. In some embodiments, anamplification reaction comprises no more than 5, 10, 15, 20, 25, 35, 50,or more cycles. Cycles can contain any number of steps, such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more steps. Steps can comprise any temperatureor gradient of temperatures, suitable for achieving the purpose of thegiven step, including but not limited to, 3′ end extension (e.g. adaptorfill-in), primer annealing, primer extension, and strand denaturation.Steps can be of any duration, including but not limited to about, lessthan about, or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 70, 80, 90, 100, 120, 180, 240, 300, 360, 420, 480, 540, 600, ormore seconds, including indefinitely until manually interrupted. Cyclesof any number comprising different steps can be combined in any order.In some embodiments, different cycles comprising different steps arecombined such that the total number of cycles in the combination isabout, less that about, or more than about 5, 10, 15, 20, 25, 30, 35,50, or more cycles. Amplification can be performed at any point during amulti reaction procedure using the methods and compositions of theinvention, e.g. before or after pooling of sequencing libraries fromindependent reaction volumes and may be used to amplify any suitabletarget molecule described herein.

Ligation Reactions

In some embodiments, the oligonucleotides can be ligated or linked toadaptors or barcodes. The linking agent can be a ligase. In someembodiments the ligase is T4 DNA ligase, using well known procedures(Maniatis, T. in Molecular Cloning, Cold Spring Harbor Laboratory(1982)). Other DNA ligases may also be used. With regard to ligation,other ligases, such as those derived from thermophilic organisms may beused thus permitting ligation at higher temperatures allowing the use oflonger oligonucleotides (with increased specificity) which could beannealed and ligated simultaneously under the higher temperaturesnormally permissible for annealing such oligonucleotides.

The terms “joining” and “ligation” as used herein, with respect to twopolynucleotides, refers to the covalent attachment of two separatepolynucleotides to produce a single larger polynucleotide with acontiguous backbone. Methods for joining two polynucleotides are knownin the art, and include without limitation, enzymatic and non-enzymatic(e.g. chemical) methods. Examples of ligation reactions that arenon-enzymatic include the non-enzymatic ligation techniques described inU.S. Pat. Nos. 5,780,613 and 5,476,930, which are herein incorporated byreference. In some embodiments, an adaptor oligonucleotide is joined toa target polynucleotide by a ligase, for example a DNA ligase or RNAligase. Multiple ligases, each having characterized reaction conditions,are known in the art, and include, without limitation NAD⁺-dependentligases including tRNA ligase, Taq DNA ligase, Thermus filiformis DNAligase, Escherichia coli DNA ligase, Tth DNA ligase, Thermus scotoductusDNA ligase (I and II), thermostable ligase, Ampligase thermostable DNAligase, VanC-type ligase, 9° N DNA Ligase, Tsp DNA ligase, and novelligases discovered by bioprospecting; ATP-dependent ligases including T4RNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Pfu DNA ligase,DNA ligase 1, DNA ligase III, DNA ligase IV, and novel ligasesdiscovered by bioprospecting; and wild-type, mutant isoforms, andgenetically engineered variants thereof. Ligation can be betweenpolynucleotides having hybridizable sequences, such as complementaryoverhangs. Ligation can also be between two blunt ends. Generally, a 5′phosphate is utilized in a ligation reaction. The 5′ phosphate can beprovided by the target polynucleotide, the adaptor oligonucleotide, orboth. 5′ phosphates can be added to or removed from polynucleotides tobe joined, as needed. Methods for the addition or removal of 5′phosphates are known in the art, and include without limitationenzymatic and chemical processes. Enzymes useful in the addition and/orremoval of 5′ phosphates include kinases, phosphatases, and polymerases.In some embodiments, both of the two ends joined in a ligation reaction(e.g. an adaptor end and a target polynucleotide end) provide a 5′phosphate, such that two covalent linkages are made in joining the twoends. In some embodiments, only one of the two ends joined in a ligationreaction (e.g. only one of an adaptor end and a target polynucleotideend) provides a 5′ phosphate, such that only one covalent linkage ismade in joining the two ends. In some embodiments, only one strand atone or both ends of a target polynucleotide is joined to an adaptoroligonucleotide. In some embodiments, both strands at one or both endsof a target polynucleotide are joined to an adaptor oligonucleotide. Insome embodiments, 3′ phosphates are removed prior to ligation. In someembodiments, an adaptor oligonucleotide is added to both ends of atarget polynucleotide, wherein one or both strands at each end arejoined to one or more adaptor oligonucleotides. When both strands atboth ends are joined to an adaptor oligonucleotide, joining can befollowed by a cleavage reaction that leaves a 5′ overhang that can serveas a template for the extension of the corresponding 3′ end, which 3′end may or may not include one or more nucleotides derived from theadaptor oligonucleotide. In some embodiments, a target polynucleotide isjoined to a first adaptor oligonucleotide on one end and a secondadaptor oligonucleotide on the other end. In some embodiments, thetarget polynucleotide and the adaptor to which it is joined compriseblunt ends. In some embodiments, separate ligation reactions are carriedout for each sample, using a different first adaptor oligonucleotidecomprising at least one barcode sequence for each sample, such that nobarcode sequence is joined to the target polynucleotides of more thanone sample. A target polynucleotide that has an adaptor/primeroligonucleotide joined to it is considered “tagged” by the joinedadaptor.

In some embodiments, nucleic acids described herein are linked makinguse of CLICK chemistry. Suitable methods to link various molecules usingCLICK chemistry are known in the art (for CLICK chemistry linkage ofoligonucleotides, see, e.g. El-Sagheer et al. (PNAS, 108:28,11338-11343, 2011). Click chemistry may be performed in the presence ofCu1.

Barcodes

Barcodes are typically known nucleic acid sequences that allow somefeature of a polynucleotide with which the barcode is associated to beidentified. In some embodiments, a barcode comprises a nucleic acidsequence that when joined to a target polynucleotide serves as anidentifier of the sample from which the target polynucleotide wasderived.

Barcodes can be designed at suitable lengths to allow sufficient degreeof identification, e.g. at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, or more nucleotides in length. Multiplebarcodes, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more barcodes, may beused on the same molecule, optionally separated by non-barcodesequences. In some embodiments, barcodes are shorter than 10, 9, 8, 7,6, 5, or 4 nucleotides in length. In some embodiments, barcodesassociated with some polynucleotides are of different length thanbarcodes associated with other polynucleotides. In general, barcodes areof sufficient length and comprise sequences that are sufficientlydifferent to allow the identification of samples based on barcodes withwhich they are associated. In some embodiments, a barcode, and thesample source with which it is associated, can be identified accuratelyafter the mutation, insertion, or deletion of one or more nucleotides inthe barcode sequence, such as the mutation, insertion, or deletion of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. In some embodiments,each barcode in a plurality of barcodes differ from every other barcodein the plurality at at least three nucleotide positions, such as atleast 3, 4, 5, 6, 7, 8, 9, 10, or more positions.

Sequencing

De novo synthesized oligonucleotide and longer polynucleotide productsdescribed herein may be subject to quality control prior to proceedingwith subsequent steps of a procedure, such as a multireaction procedure.Quality control may be applied while keeping individual products inseparate volumes, such as on resolved features of a substrate asdescribed herein. A fraction may be aliquoted for quality control, whilethe rest of the volumes compartmentalizing each product remainindividually accessible.

FIG. 17 illustrates an example quality control procedure comprising nextgeneration sequencing. Gene specific padlock probes targeting a specificproduct are designed to cover overlapping sequence segments of theproduct that is being tested. The ends of the individual padlock probesspecific for a gene product may be designed to be hybridizable toregions scattered along the gene product for proper coverage duringsequencing. All probes specific for the same gene product may comprise abarcode sequence associated with that gene product. A suitablepolymerase and/or ligase may be used to fill between the ends of thepadlock probes along the gene product target. In some cases, the padlockprobes will form circular single stranded DNA. The typically linear geneproduct may be digested, for example after aliquoting a fraction of thegene product volume. Alternatively, a fraction of the gene productvolume may be aliquoted prior to the addition of padlock probes. Thepadlock probes carrying segments of the gene product may be amplified,e.g. using PCR. Universal or specific primer binding regions on thepadlock probes may be targeted during amplification. Sequencing primerbinding regions may be originally present in the padlock probes or maybe added during subsequent steps, e.g. by utilizing sequencing adaptorsprior to, during, or after amplification.

In various embodiments, the gene product specific padlock probes will bepooled after the initial sequencing library steps. In those cases, thegene product specific barcodes may be utilized to track sequenceinformation back to the individual gene products. The sequencinginformation obtained by any suitable means described herein or otherwiseknown in the art may be deconvoluted, e.g. by binning into individualsequence pool based on the barcode information. Suitable alignment andsequence confirmation algorithms known in the art can be utilized tofinalize quality control. Error rates and locations can be analyzed bysequence locus, by gene product, by library, or by library subsegment.The error analysis may inform acceptance or rejection of products forsubsequent steps or for delivery to a requester.

In any of the embodiments, the detection or quantification analysis ofthe oligonucleotides can be accomplished by sequencing. The subunits orentire synthesized oligonucleotides can be detected via full sequencingof all oligonucleotides by any suitable methods known in the art, e.g.,Illumina HiSeq 2500, including the sequencing methods described herein.

Sequencing can be accomplished through classic Sanger sequencing methodswhich are well known in the art. Sequencing can also be accomplishedusing high-throughput systems some of which allow detection of asequenced nucleotide immediately after or upon its incorporation into agrowing strand, i.e., detection of sequence in red time or substantiallyreal time. In some cases, high throughput sequencing generates at least1,000, at least 5,000, at least 10,000, at least 20,000, at least30,000, at least 40,000, at least 50,000, at least 100,000 or at least500,000 sequence reads per hour; with each read being at least 50, atleast 60, at least 70, at least 80, at least 90, at least 100, at least120 or at least 150 bases per read.

In some embodiments, high-throughput sequencing involves the use oftechnology available by Illumina's Genome Analyzer IIX, MiSeq personalsequencer, or HiSeq systems, such as those using HiSeq 2500, HiSeq 1500,HiSeq 2000, or HiSeq 1000. These machines use reversibleterminator-based sequencing by synthesis chemistry. These machines cando 200 billion DNA or more reads in eight days. Smaller systems may beutilized for runs within 3, 2, 1 days or less time. Short synthesiscycles may be used to minimize the time it takes to obtain sequencingresults.

In some embodiments, high-throughput sequencing involves the use oftechnology available by ABI Solid System. This genetic analysis platformthat enables massively parallel sequencing of clonally-amplified DNAfragments linked to beads. The sequencing methodology is based onsequential ligation with dye-labeled oligonucleotides.

The next generation sequencing can comprise ion semiconductor sequencing(e.g., using technology from Life Technologies (Ion Torrent)). Ionsemiconductor sequencing can take advantage of the fact that when anucleotide is incorporated into a strand of DNA, an ion can be released.To perform ion semiconductor sequencing, a high density array ofmicromachined wells can be formed. Each well can hold a single DNAtemplate. Beneath the well can be an ion sensitive layer, and beneaththe ion sensitive layer can be an ion sensor. When a nucleotide is addedto a DNA, H+ can be released, which can be measured as a change in pH.The H+ ion can be converted to voltage and recorded by the semiconductorsensor. An array chip can be sequentially flooded with one nucleotideafter another. No scanning, light, or cameras can be required. In somecases, an IONPROTON™ Sequencer is used to sequence nucleic acid. In somecases, an IONPGM™ Sequencer is used. The Ion Torrent Personal GenomeMachine (PGM) can do 10 million reads in two hours.

In some embodiments, high-throughput sequencing involves the use oftechnology available by Helicos BioSciences Corporation (Cambridge,Mass.) such as the Single Molecule Sequencing by Synthesis (SMSS)method. SMSS is unique because it allows for sequencing the entire humangenome in up to 24 hours. Finally, SMSS is powerful because, like theMIP technology, it does not require a pre amplification step prior tohybridization. In fact, SMSS does not require any amplification. SMSS isdescribed in part in US Publication Application Nos. 2006002471 I;20060024678; 20060012793; 20060012784; and 20050100932.

In some embodiments, high-throughput sequencing involves the use oftechnology available by 454 Lifesciences, Inc. (Branford, Conn.) such asthe Pico Titer Plate device which includes a fiber optic plate thattransmits chemiluninescent signal generated by the sequencing reactionto be recorded by a CCD camera in the instrument. This use of fiberoptics allows for the detection of a minimum of 20 million base pairs in4.5 hours.

Methods for using bead amplification followed by fiber optics detectionare described in Marguiles, M., et al. “Genome sequencing inmicrofabricated high-density picolitre reactors”, Nature, doi:10.1038/nature03959; and well as in US Publication Application Nos.20020012930; 20030058629; 20030100102; 20030148344; 20040248161;20050079510, 20050124022; and 20060078909.

In some embodiments, high-throughput sequencing is performed usingClonal Single Molecule Array (Solexa, Inc.) or sequencing-by-synthesis(SBS) utilizing reversible terminator chemistry. These technologies aredescribed in part in U.S. Pat. Nos. 6,969,488; 6,897,023; 6,833,246;6,787,308; and US Publication Application Nos. 20040106130; 20030064398;20030022207; and Constans, A., The Scientist 2003, 17(13):36.High-throughput sequencing of oligonucleotides can be achieved using anysuitable sequencing method known in the art, such as thosecommercialized by Pacific Biosciences, Complete Genomics, GeniaTechnologies, Halcyon Molecular, Oxford Nanopore Technologies and thelike. Other high-throughput sequencing systems include those disclosedin Venter, J., et al. Science 16 February 2001; Adams, M. et al, Science24 March 2000; and M. J, Levene, et al. Science 299:682-686, January2003; as well as US Publication Application No. 20030044781 and2006/0078937. Overall such systems involve sequencing a targetoligonucleotide molecule having a plurality of bases by the temporaladdition of bases via a polymerization reaction that is measured on amolecule of oligonucleotide, i e., the activity of a nucleic acidpolymerizing enzyme on the template oligonucleotide molecule to besequenced is followed in real time. Sequence can then be deduced byidentifying which base is being incorporated into the growingcomplementary strand of the target oligonucleotide by the catalyticactivity of the nucleic acid polymerizing enzyme at each step in thesequence of base additions. A polymerase on the target oligonucleotidemolecule complex is provided in a position suitable to move along thetarget oligonucleotide molecule and extend the oligonucleotide primer atan active site. A plurality of labeled types of nucleotide analogs areprovided proximate to the active site, with each distinguishably type ofnucleotide analog being complementary to a different nucleotide in thetarget oligonucleotide sequence. The growing oligonucleotide strand isextended by using the polymerase to add a nucleotide analog to theoligonucleotide strand at the active site, where the nucleotide analogbeing added is complementary to the nucleotide of the targetoligonucleotide at the active site. The nucleotide analog added to theoligonucleotide primer as a result of the polymerizing step isidentified. The steps of providing labeled nucleotide analogs,polymerizing the growing oligonucleotide strand, and identifying theadded nucleotide analog are repeated so that the oligonucleotide strandis further extended and the sequence of the target oligonucleotide isdetermined.

The next generation sequencing technique can comprises real-time (SMRT™)technology by Pacific Biosciences. In SMRT, each of four DNA bases canbe attached to one of four different fluorescent dyes. These dyes can bephospho linked. A single DNA polymerase can be immobilized with a singlemolecule of template single stranded DNA at the bottom of a zero-modewaveguide (ZMW). A ZMW can be a confinement structure which enablesobservation of incorporation of a single nucleotide by DNA polymeraseagainst the background of fluorescent nucleotides that can rapidlydiffuse in an out of the ZMW (in microseconds). It can take severalmilliseconds to incorporate a nucleotide into a growing strand. Duringthis time, the fluorescent label can be excited and produce afluorescent signal, and the fluorescent tag can be cleaved off. The ZMWcan be illuminated from below. Attenuated light from an excitation beamcan penetrate the lower 20-30 nm of each ZMW. A microscope with adetection limit of 20 zepto liters (10″ liters) can be created. The tinydetection volume can provide 1000-fold improvement in the reduction ofbackground noise. Detection of the corresponding fluorescence of the dyecan indicate which base was incorporated. The process can be repeated.

In some cases, the next generation sequencing is nanopore sequencing{See e.g., Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001). Ananopore can be a small hole, of the order of about one nanometer indiameter Immersion of a nanopore in a conducting fluid and applicationof a potential across it can result in a slight electrical current dueto conduction of ions through the nanopore. The amount of current whichflows can be sensitive to the size of the nanopore. As a DNA moleculepasses through a nanopore, each nucleotide on the DNA molecule canobstruct the nanopore to a different degree. Thus, the change in thecurrent passing through the nanopore as the DNA molecule passes throughthe nanopore can represent a reading of the DNA sequence. The nanoporesequencing technology can be from Oxford Nanopore Technologies; e.g., aGridlON system. A single nanopore can be inserted in a polymer membraneacross the top of a microwell. Each microwell can have an electrode forindividual sensing. The microwells can be fabricated into an array chip,with 100,000 or more microwells (e.g., more than 200,000, 300,000,400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000) perchip. An instrument (or node) can be used to analyze the chip. Data canbe analyzed in real-time. One or more instruments can be operated at atime. The nanopore can be a protein nanopore, e.g., the proteinalpha-hemolysin, a heptameric protein pore. The nanopore can be asolid-state nanopore made, e.g., a nanometer sized hole formed in asynthetic membrane (e.g., SiNx, or SiO2). The nanopore can be a hybridpore (e.g., an integration of a protein pore into a solid-statemembrane). The nanopore can be a nanopore with an integrated sensors(e.g., tunneling electrode detectors, capacitive detectors, or graphenebased nano-gap or edge state detectors (see e.g., Garaj et al. (2010)Nature vol. 67, doi: 10.1038/nature09379)). A nanopore can befunctionalized for analyzing a specific type of molecule (e.g., DNA,RNA, or protein). Nanopore sequencing can comprise “strand sequencing”in which intact DNA polymers can be passed through a protein nanoporewith sequencing in real time as the DNA translocates the pore. An enzymecan separate strands of a double stranded DNA and feed a strand througha nanopore. The DNA can have a hairpin at one end, and the system canread both strands. In some cases, nanopore sequencing is “exonucleasesequencing” in which individual nucleotides can be cleaved from a DNAstrand by a processive exonuclease, and the nucleotides can be passedthrough a protein nanopore. The nucleotides can transiently bind to amolecule in the pore (e.g., cyclodextran). A characteristic disruptionin current can be used to identify bases.

Nanopore sequencing technology from GENIA can be used. An engineeredprotein pore can be embedded in a lipid bilayer membrane. “ActiveControl” technology can be used to enable efficient nanopore-membraneassembly and control of DNA movement through the channel. In some cases,the nanopore sequencing technology is from NABsys. Genomic DNA can befragmented into strands of average length of about 100 kb. The 100 kbfragments can be made single stranded and subsequently hybridized with a6-mer probe. The genomic fragments with probes can be driven through ananopore, which can create a current-versus-time tracing. The currenttracing can provide the positions of the probes on each genomicfragment. The genomic fragments can be lined up to create a probe mapfor the genome. The process can be done in parallel for a library ofprobes. A genome-length probe map for each probe can be generated.Errors can be fixed with a process termed “moving window Sequencing ByHybridization (mwSBH).” In some cases, the nanopore sequencingtechnology is from IBM/Roche. An electron beam can be used to make ananopore sized opening in a microchip. An electrical field can be usedto pull or thread DNA through the nanopore. A DNA transistor device inthe nanopore can comprise alternating nanometer sized layers of metaland dielectric. Discrete charges in the DNA backbone can get trapped byelectrical fields inside the DNA nanopore. Turning off and on gatevoltages can allow the DNA sequence to be read.

The next generation sequencing can comprise DNA nanoball sequencing (asperformed, e.g., by Complete Genomics; see e.g., Drmanac et al. (2010)Science 327: 78-81). DNA can be isolated, fragmented, and size selected.For example, DNA can be fragmented (e.g., by sonication) to a meanlength of about 500 bp. Adaptors (Ad1) can be attached to the ends ofthe fragments. The adaptors can be used to hybridize to anchors forsequencing reactions. DNA with adaptors bound to each end can be PCRamplified. The adaptor sequences can be modified so that complementarysingle strand ends bind to each other forming circular DNA. The DNA canbe methylated to protect it from cleavage by a type IIS restrictionenzyme used in a subsequent step. An adaptor (e.g., the right adaptor)can have a restriction recognition site, and the restriction recognitionsite can remain non-methylated. The non-methylated restrictionrecognition site in the adaptor can be recognized by a restrictionenzyme (e.g., AcuI), and the DNA can be cleaved by AcuI 13 bp to theright of the right adaptor to form linear double stranded DNA. A secondround of right and left adaptors (Ad2) can be ligated onto either end ofthe linear DNA, and all DNA with both adapters bound can be PCRamplified (e.g., by PCR). Ad2 sequences can be modified to allow them tobind each other and form circular DNA. The DNA can be methylated, but arestriction enzyme recognition site can remain non-methylated on theleft Adl adapter. A restriction enzyme (e.g., AcuI) can be applied, andthe DNA can be cleaved 13 bp to the left of the Adl to form a linear DNAfragment. A third round of right and left adaptor (Ad3) can be ligatedto the right and left flank of the linear DNA, and the resultingfragment can be PCR amplified. The adaptors can be modified so that theycan bind to each other and form circular DNA. A type III restrictionenzyme (e.g., EcoP15) can be added; EcoP15 can cleave the DNA 26 bp tothe left of Ad3 and 26 bp to the right of Ad2. This cleavage can removea large segment of DNA and linearize the DNA once again. A fourth roundof right and left adaptors (Ad4) can be ligated to the DNA, the DNA canbe amplified (e.g., by PCR), and modified so that they bind each otherand form the completed circular DNA template.

Rolling circle replication (e.g., using Phi 29 DNA polymerase) can beused to amplify small fragments of DNA. The four adaptor sequences cancontain palindromic sequences that can hybridize and a single strand canfold onto itself to form a DNA nanoball (DNB™) which can beapproximately 200-300 nanometers in diameter on average. A DNA nanoballcan be attached (e.g., by adsorption) to a microarray (sequencingflowcell). The flow cell can be a silicon wafer coated with silicondioxide, titanium and hexamehtyldisilazane (HMDS) and a photoresistmaterial. Sequencing can be performed by unchained sequencing byligating fluorescent probes to the DNA. The color of the fluorescence ofan interrogated position can be visualized by a high resolution camera.The identity of nucleotide sequences between adaptor sequences can bedetermined.

Inkjet Deposits

The methods and compositions of the invention, in some embodiments, makeuse of depositing, positioning, or placing a composition at a specificlocation on or in the surface of a support. Depositing may comprisecontacting one composition with another. Depositing may be manual orautomatic, e.g., depositing may be accomplished by automated roboticdevices. Pulse jets or inkjets may be used to dispense drops of a fluidcomposition onto a support. Pulse jets typically operate by delivering apulse of pressure (such as by a piezoelectric or thermoelectric element)to liquid adjacent to an outlet or orifice such that a drop can bedispensed therefrom.

Liquids of reagents can be deposited to resolved loci of a substratedescribed in further detail elsewhere herein using various methods orsystems known in the art. Microdroplets of fluid can be delivered to asurface or resolved loci on or within a substrate described in thecurrent invention at submicron precision. Commercially availabledispensing equipments using inkjet technology as the microdispensingmethod for fluid volume below can be employed. The droplets producedusing ink-jet technology are highly reproducible and can be controlledso that a droplet may be placed on a specific location at a specifictime according to digitally stored image data. Typical droplet diametersfor demand mode ink-jet devices can be 30-100 μm, which translates todroplet volumes of 14-520 pl. Droplet creation rates for demand modeink-jet devices can be 2000-5000 droplets per second. Demand modeink-jet microdispensing can be utilized at suitable resolutions andthroughputs to service substrates with high densities of resolved locidescribed in further detail elsewhere herein. Methods and systems fordepositing or delivering reagents are described in further detail inU.S. Pat. Nos. 5,843,767 and 6,893,816, both of which are incorporatedby reference in their entirety.

The systems for depositing or delivering the reagents to resolved locican comprise one or more subsystems including but not limited to: amicrojet dispense head, a fluid delivery system or an inkjet pump, a X-Ypositioning system, a vision system, or a system controller. Themicrojet dispense head can be an assembly of a plurality of MicroJetdevices (e.g., 8 MicroJet devices) and the required drive electronics.The system complexity can be minimized by using a single channel ofdrive electronics to multiplex the 8 or 10 dispensing devices. Drivewaveform requirements for each individual device can be downloaded fromthe system controller. The drive electronics can be constructed usingconventional methods that are known in the art. The fluid deliverysystem, or the inkjet pump, can be a Beckman Biomec that is modified toact as the multiple reagent input system. Between it and the MicroJetdispense head can be a system of solenoid valves, controlled by thesystem controller. They provide pressurized flushing fluid and air topurge reagent from the system and vacuum to load reagent into thesystem. The X-Y positioning system can be any commercially availableprecision X-Y positioning system with a controller. The positioningsystem can be sized to accommodate a plurality of sensors. The visionsystem can be used to calibrate the “landing zone” of each MicroJetdevice relative to the positioning system. Calibration may occur aftereach reagent loading cycle. Also, the vision system can locate eachdispensing site on each sensor when the sensor tray is first loaded viafiducial marks on the sensors. A software based system or a hardwarebased vision system can be used. The system controller can be a standardcomputer system that is used as the overall system controller. Thevision system image capture and processing also reside on the systemcontroller. Systems for depositing or delivering the reagents toresolved loci are described in further detail in PCT Pub. No.WO2000039344, which is incorporated herein by reference in its entirety.

FIG. 18 illustrates an example of an inkjet assembly. In someembodiments, the inkjet assembly can comprise at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 32, 34, 36, 38, 40, 45, 48, 50, 56, 60, 64, 72, 75,80, 85, 90, 95, 100 or more inkjet heads. The inkjets heads may eachdeposit a different codon (trinucleotide) building blocks. In anexemplary embodiment, inkjet heads can have Silicon orifice plates with256 nozzles on 254 μm centers and 100 μm fly height. Each head can haveaccess to each well that traverses. The inkjet assembly can have a scanspeed about 100 mm/s with precision in the traveling (x,y) plane that isabout 2 μm. In some cases, the scan height over wafer of the inkjetassembly can be about 100 μm with a flatness runout of about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μm. In somecases, the inkjet assembly can comprise a vision system to align inkjetwith substrates, e.g. silicon wafers, chucked on a vacuum chuck, in somecases as part of a flowcell assembly.

In some cases, methods and systems of depositing reagents to a pluralityof resolved loci described herein can comprise applying through aninkjet pump at least one microdrop of a first reagent to a first locusof the plurality of loci and applying through an inkjet pump at leastone microdrop of a second reagent to a second locus of the plurality ofresolved loci. In some embodiments, the second locus can be adjacent tothe first locus, and the first and second reagents can be different. Thefirst and second loci can reside on microstructures fabricated into asupport surface and the microstructures can comprise at least onechannel. In some cases, the at least one channel is more than 100 μmdeep. In some embodiments, the first and the second reagents can be thesame. In some cases, the microstructures comprise a large microchanneland one or more microchannels that are fluidically connected to thefirst microchannel. The large initial microchannel initially receives adeposited liquid, typically reducing any cross contamination of reagentsto and from adjacent microstructures. The contents of the droplet cansubsequently flow into the one or more smaller microchannels, which mayhost suitable surfaces for the reactions described herein, such asoligonucleotide synthesis.

The at least one channel can have a depth that can be about, at leastabout, or less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000μm. In some embodiments, the at least one channel can have a depth thatcan be between about 50-100, 50-150, 50-200, 100-200, 100-300, 20-300 or20-100 μm. In some embodiments, the at least one channel can be morethan 100 μm deep.

Each of the droplets of reagents can have a suitable volume that cantraverse through the depth of the microchannel without losing momentum.The suitable volume can comprise a desired amount of reagents foroligonucleotide synthesis. For example, without limitation, each of thedroplets comprising reagents can have a volume that is about or at leastabout 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 400, 500 pl, 1, 1.5, 2, 2.5, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 200, 500 nl, or more.In various embodiments, the system is adjusted such that any satellitedroplets trailing a deposited droplet is small enough to minimizecross-contamination. In the case of an inkjet, the printheads can bebrought sufficiently close to a substrate e.g. within 100 μm, such thata deposited droplet and its satellite drops are substantially within achannel of the substrate before aerosol movement. The satellite dropletsmay have a diameter of less than 0.5, 1, 1.5 or 2 μm. In variousembodiments, the by volume fraction of satellite droplets that engage inaerosol movement is less than 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01% of adeposited droplet, or less.

As described elsewhere herein, the microstructures can comprise multiplechannels in fluidic communication with each other. In some cases, themicrostructures can comprise at least three, four, five, six, seven,eight, nine or ten channels in fluid communications. The channels canhave different dimensions, e.g. widths or lengths, as described infurther detail elsewhere herein. In some embodiments, the fluidicallyconnected channels of the microstructures can comprise two or morechannels with the same width, length, and/or other dimensions.

The microdroplets of fluid can be delivered to a surface or resolvedloci within a substrate as described elsewhere herein at a highprecision with minimal cross-contamination. In some cases, the firstlocus can receive less than 0.1% of a second reagent that is intended tobe deposited to a second locus and similarly the second locus canreceive less than 0.1% of the first reagent. In some cases, the firstlocus can receive less than about 0.5%, 0.45%, 0.4%, 0.35%, 0.3%, 0.25%,0.2%, 0.15%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02% or 0.01% of the secondreagent. The second locus can receive less than about 0.5%, 0.45%, 0.4%,0.35%, 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02% or0.01% of the first reagent.

In some cases, the reagents can be delivered in droplets that have adiameter of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190 or 200 μm. The droplets of reagent can have adiameter that is at least about 2 μm. The reagents can be delivered indroplets that have a diameter of less than about 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200μm. The reagents can be delivered in droplets that have a diameter ofbetween 2-10, 2-5, 10-200, 10-150, 10-100, 10-500, 20-200, 20-150,20-100, 30-100, 30-200, 30-150, 40-100, 40-80 or 50-60 μm.

The droplets of reagents can be deposited in a rate of about or at leastabout 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 dropletsper second.

Soft Landing

Systems and methods for depositing droplets to a plurality of microwellsare also described herein. In one aspect, droplets can be deposited intoa microwell of a microfluidic system comprising a first surface with aplurality of microwells. The droplet can have a suitable Reynoldsnumber, such as about 1-1000, 1-2000, 1-3000, 0.5-1000, 0.5-2000,0.5-3000, 0.5-4000, 0.5-5000, 1-500, 2-500, 1-100, 2-100, 5-100, 1-50,2-50, 5-50 or 10-50, such that bouncing of liquids is minimized uponreaching the bottom of the microwell. Those of skill in the artappreciate that the Reynolds number may fall within any range bounded byany of these values (e.g., about 0.5 to about 500). Suitable methods foraccurate estimation of Reynolds numbers in fluid systems are describedin Clift et al. (Clift, Roland, John R. Grace, and Martin E. Weber,Bubbles, Drops and Particles, 2005. Dover Publications) and Happel etal. (Happel, John and Howard Brenner, 1965. Prentice-Hall), both ofwhich are herein incorporated by reference in their entirety.

The density of the plurality of microwells can be more than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 1000 ormore per mm². Following the methods described herein, the droplet of theliquid can flow through the microwell smoothly and land on the bottom ofthe microwell softly.

The liquid droplets can be deposited using any methods and systems knownin the art. In some embodiments, the microfluidic system can furthercomprise an inkjet pump. The inkjet pump can be used to deposit theliquid droplet to one of the plurality of microwells. Variousembodiments of the liquid deposit systems are described elsewhere in thespecification.

In some cases, the microwells can be in different width, the same width,or a combination of the same or different width within subregions of asubstrate. The microwells can have any different width. For example,without limitation, the width of the microwells can be about, wider thanabout, or narrower than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μm.

The microwells can have any different length. For example, withoutlimitation, the length of the microwells can be about, longer thanabout, or shorter than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 or1000 μm.

The microwells can be fluidically connected to at least onemicrochannel. The microwells can comprise a ratio of surface area tolength, or a perimeter, of about, at least about, or less than about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 or 100 μm.

The droplets of the liquid can have a volume that is suitable for themethods described herein. In some embodiments, the droplet can have avolume that is less than about 0.5 microliters (μl), less than about 1μl, less than about 1.5 μl, less than about 2 μl, less than about 2.5μl, less than about 3 μl, less than about 3.5 μl, less than about 4 μl,less than about 4.5 μl, less than about 5 μl, less than about 5.5 μl,less than about 6 μl, less than about 6.5 μl, less than about 7 μl, lessthan about 7.5 μl, less than about 8 μl, less than about 8.5 μl, lessthan about 9 μl, less than about 9.5 μl, less than about 10 μl, lessthan about 11 μl, less than about 12 μl, less than about 13 μl, lessthan about 14 μl, less than about 15 μl, less than about 16 μl, lessthan about 17 μl, less than about 18 μl, less than about 19 μl, lessthan about 20 μl, less than about 25 μl, less than about 30 μl, lessthan about 35 μl, less than about 40 μl, less than about 45 μl, lessthan about 50 μl, less than about 55 μl, less than about 60 μl, lessthan about 65 μl, less than about 70 μl, less than about 75 μl, lessthan about 80 μl, less than about 85 μl, less than about 90 μl, lessthan about 95 μl or less than about 100 μl. In some embodiments, thedroplet can have a volume that is about 0.5 microliters (μl), about 1μl, about 1.5 μl, about 2 μl, about 2.5 μl, about 3 μl, about 3.5 μl,about 4 μl, about 4.5 μl, about 5 μl, about 5.5 μl, about 6 μl, about6.5 μl, about 7 μl, about 7.5 μl, about 8 μl, about 8.5 μl, about 9 μl,about 9.5 μl, about 10 μl, about 11 μl, about 12 μl, about 13 μl, about14 μl, about 15 μl, about 16 μl, about 17 μl, about 18 μl, about 19 μl,about 20 μl, about 25 μl, about 30 μl, about 35 μl, about 40 μl, about45 μl, about 50 μl, about 55 μl, about 60 μl, about 65 μl, about 70 μl,about 75 μl, about 80 μl, about 85 μl, about 90 μl, about 95 μl or about100 μl.

In some cases, the microchannels can be coated with a moiety, such as achemically inert moiety, that increases surface energy. The types ofsuitable chemically inert or reactive moieties are described elsewherein the current specification.

The Reynolds number of the droplet can be at a range of Reynolds numberthat allows the liquid to flow smoothly through microwells and/ormicrochannels as described herein. In some embodiments, the Reynoldsnumber of the droplet can be less than about 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, or 1000. In some embodiments, the Reynolds number of the dropletcan be more than about 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or1000. In some cases, the droplets can flow through the microwells in alaminar flow or near-laminar flow.

The droplet can be applied or deposited at a velocity of at least 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 m/sec or higher.

Programmable Split

The system as described herein can comprise a plurality of resolved lociand a plurality of resolved reactor caps that can be sealed together toform a plurality of resolved reactors. The plurality of resolvedreactors can contain reagents. The sealing may be reversible or loose,and the plurality of resolved reactor caps can be released from theplurality of resolved loci. Upon release from the first surfacecomprising the plurality of resolved loci, the reactor caps can retainat least a portion of the reagents. By controlling the release of thereactor caps from the plurality of resolved loci, the partitioning ofthe liquid or the reagents can be controlled. In one aspect of theinstant invention, a method of partitioning is described herein. Themethod may comprise contacting a first surface comprising a liquid at afirst plurality of resolved loci with a second surface comprising asecond plurality of resolved loci, such as reactor caps, wherein thefirst surface can comprise a first surface tension with the liquid, thesecond surface can comprise a second surface tension with the liquid anddetermining a velocity of release such that a desired fraction of theliquid can be transferred from the first plurality of resolved loci tothe second plurality of resolved loci Upon detaching the second surfacefrom the first surface at this calculated velocity, a desired fractionof the contents of the reactors may be retained in reactors. The firstsurface comprising the first plurality of resolved loci may comprise theplurality of resolved loci that are coated with oligonucleotides. Thesecond surface comprising the second plurality of resolved loci may be acapping element comprising a plurality of reactor caps. In some cases,the method can further comprise contacting a third surface with a thirdplurality of resolved loci. Various aspects or embodiments are describedherein.

The liquid that is retained in the second surface may be held by anymethods known in the art. In some cases, the first or the second surfacecan comprise microchannels holding at least a portion of the liquid. Insome cases, the first or the second surface can comprise nanoreactorsholding at least a portion of the liquid. In some cases, the liquid canbe retained due to the surface tension differences between the first andthe second surface. Without being bound by theory, for water basedliquids, a higher portion of the liquid may be retained on the surfacehaving higher surface energy, or less hydrophobic.

The liquid may be partitioned such that a desired fraction of thereagents can be retained onto the first or the second surface uponreleasing. For example, without limitation, the desired fraction may beabout, at least about, or more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

Parallel Microfluidic Mixing Methods

In another aspect of the current invention, methods of mixing liquid aredescribed herein. The methods can comprise providing a first substratecomprising a plurality of microstructures fabricated thereto; providinga second substrate comprising a plurality of resolved reactor caps;aligning the first and second substrates such that a first reactor capof the plurality is configured to receive liquid from n microstructuresin the first substrate; and delivering liquid from the n microstructuresinto the first reactor cap, thereby mixing liquid from the nmicrostructures forming a mixture. Various embodiments and variationsare described herein.

The density of the resolved reactor caps can be any suitable densitythat allows desired alignment of the microstructures of a firstsubstrate and the reactor caps of a second substrate. In some cases, thedensity of the resolved reactor caps can be at least 1/mm². In somecases, the density of the resolved reactors can be about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 15, about 20, about 25, about 30, about 35, about 40, about 50,about 75, about 100, about 200, about 300, about 400, about 500, about600, about 700, about 800, about 900, about 1000, about 1500, or about2000 sites per 1 mm². In some embodiments, the density of the resolvedreactors can be at least about 1, at least about 2, at least about 3, atleast about 4, at least about 5, at least about 6, at least about 7, atleast about 8, at least about 9, at least about 10, at least about 20,at least about 30, at least about 40, at least about 50, at least about75, at least about 100, at least about 200, at least about 300, at leastabout 400, at least about 500, at least about 600, at least about 700,at least about 800, at least about 900, at least about 1000, at leastabout 1500, at least about 2000, or at least about 3000 sites per 1 mm².

The microstructures can be at any density practicable according to themethods and compositions of the invention. In some cases, themicrostructures can be at a density of about, at least about, or lessthan about 1, about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 50, about 75, about 100, about 200, about 300,about 400, about 500, about 600, about 700, about 800, about 900, about1000, about 1500, about 2000, or about 3000 sites per 1 mm². In someembodiments, the microstructures can be at a density of at least 100 per1 mm². In some cases, the microstructures can have a surface densitythat is about the same as the density of the resolved reactors.

In some cases, there can be a gap, e.g. a gap of less than about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or200 μm between the first and the second substrates after aligning thefirst and the second substrates such that a first reactor cap of theplurality is configured to receive liquid from n microstructures in thefirst substrate.

In some cases, the mixture or the liquid can partially spread into thegap between the first and the second substrates after aligning the firstand the second substrates such that a first reactor cap of the pluralityis configured to receive liquid from n microstructures in the firstsubstrate. The liquid or mixture that partially spreads into the gap mayform a capillary burst valve. The methods of mixing can further comprisesealing the gap by bringing the first and the second substrate closertogether. In some cases, the first and the second substrate can be indirect physical contact.

The plurality of microstructures and reactor caps can have any suitabledesign or dimensions as described in further detail elsewhere herein. Atleast one channel can have a cross-sectional area that is in a circularshape and can comprise a radius of the cross-sectional area of about, atleast about, less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μm.

In some cases, the channels may be coated with a moiety, such as achemically inert moiety, that increases surface energy corresponding toa water contact angle of less than 90°. The surface energy, orhydrophobicity of a surface, can be evaluated or measured by measuring awater contact angle. A water contact angle of smaller than 90° mayfunctionalize the solid surface in a relatively hydrophilic manner Awater contact angle of greater than 90° may functionalize the solidsurface in a relatively hydrophobic manner Highly hydrophobic surfaceswith low surface energy can have water contact angles that are greaterthan 120°. In some cases, the surface of the channels, or one of the twochannels as described herein can be functionalized or modified to behydrophobic, to have a low surface energy, or to have a water contactangle that can be greater than about 90°, 95°, 100°, 105°, 110°, 115°,120°, 125°, 130°, 135°, 140°, 145° or 150° as measured on an uncurvedsurface. In some cases, the surface of the channels, or one of the twochannels as described herein in the current invention can befunctionalized or modified to be hydrophilic, to have a high surfaceenergy, or to have a water contact angle that can be less than about90°, 85°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15° or 10° as measured on an uncurved surface. The surface of thechannels or one of the two channels can be functionalized or modified tobe more hydrophilic or hydrophobic. In some cases, the surfaces of thefirst and the second substrate can comprise a different surface energywith a given liquid, such as water. In some cases, the surfaces of thefirst and the second substrates can comprise a differential watercontact angle of between about 5°, 10°, 20°, 30°, 40°, 50°, 60°, 70°,80°, 90°. Other methods for functionalizing the surface are described inU.S. Pat. No. 6,028,189, which is herein incorporated by reference inits entirety.

In some embodiments, the delivering can be performed by pressure. Thedelivering liquid from the n microstructures into the first reactor capcan result in mixing liquid from the n microstructures and forming amixture.

In some cases, the volume of the total mixture liquid can be greaterthan the volume of the reactor cap. All or part of the reactor capsurfaces, such as the rim surface, may be modified using suitablesurface modification methods described in further detail elsewhereherein and otherwise known in the art. In some cases, surfaceirregularities are engineered. Chemical surface modifications andirregularities may serve to adjust the water contact angle of the rim.Similar surface treatments may also be applied on the surface of asubstrate that is brought in close proximity to the reactor caps forminga seal, e.g. a reversible seal. A capillary burst valve may be utilizedbetween the two surfaces as described in further detail elsewhereherein. The surface treatments can be useful in precise control of suchseals comprising capillary burst valves.

In some cases, the releasing of the capping element from the firstsurface, and the releasing of the capping element from the secondsurface can be performed at a different velocity. The amount of theportion of reagents that is retained upon releasing the capping elementfrom the corresponding surface can be controlled by the velocity or thesurface energy of the capping element and the corresponding surface. Thedifference in the surface energy, or hydrophobicity, of the cappingelement and the corresponding surface can be a parameter to control theportion of the reagents that is retained upon release. The volume of thefirst and the second reactions can be different.

Downstream Applications

The methods and compositions of the invention may be used for nucleicacid hybridization studies such as gene expression analysis, genotyping,heteroduplex analysis, nucleic acid sequencing determinations based onhybridization, synthesis of DNA, RNA, peptides, proteins or otheroligomeric or non-oligomeric molecules, combinatorial libraries forevaluation of candidate drugs.

DNA and RNA synthesized in accordance with the invention may be used inany application including, by way of example, probes for hybridizationmethods such as gene expression analysis, genotyping by hybridization(competitive hybridization and heteroduplex analysis), sequencing byhybridization, probes for Southern blot analysis (labeled primers),probes for array (either microarray or filter array) hybridization,“padlock” probes usable with energy transfer dyes to detecthybridization in genotyping or expression assays, and other types ofprobes. The DNA and RNA prepared in accordance with the invention mayalso be used in enzyme-based reactions such as polymerase chain reaction(PCR), as primers for PCR, templates for PCR, allele-specific PCR(genotyping/haplotyping) techniques, real-time PCR, quantitative PCR,reverse transcriptase PCR, and other PCR techniques. The DNA and RNA maybe used for various ligation techniques, including ligation-basedgenotyping, oligo ligation assays (OLA), ligation-based amplification,ligation of adapter sequences for cloning experiments, Sanger dideoxysequencing (primers, labeled primers), high throughput sequencing (usingelectrophoretic separation or other separation method), primerextensions, mini-sequencings, and single base extensions (SBE). The DNAand RNA produced in accordance with the invention may be used inmutagenesis studies, (introducing a mutation into a known sequence withan oligo), reverse transcription (making a cDNA copy of an RNAtranscript), gene synthesis, introduction of restriction sites (a formof mutagenesis), protein-DNA binding studies, and like experiments.Various other uses of DNA and RNA produced by the subject methods willsuggest themselves to those skilled in the art, and such uses are alsoconsidered to be within the scope of this disclosure.

Computer Systems

In various embodiments, the methods and systems of the invention mayfurther comprise software programs on computer systems and use thereof.Accordingly, computerized control for the synchronization of thedispense/vacuum/refill functions such as orchestrating and synchronizingthe printhead movement, dispense action and vacuum actuation are withinthe bounds of the invention. The computer systems may be programmed tointerface between the user specified base sequence and the position of adispenser head to deliver the correct reagents to specified regions ofthe substrate.

The computer system 1900 illustrated in FIG. 19 may be understood as alogical apparatus that can read instructions from media 1911 and/or anetwork port 1905, which can optionally be connected to server 1909having fixed media 1912. The system, such as shown in FIG. 19 caninclude a CPU 1901, disk drives 1903, optional input devices such askeyboard 1915 and/or mouse 1916 and optional monitor 1907. Datacommunication can be achieved through the indicated communication mediumto a server at a local or a remote location. The communication mediumcan include any means of transmitting and/or receiving data. Forexample, the communication medium can be a network connection, awireless connection or an internet connection. Such a connection canprovide for communication over the World Wide Web. It is envisioned thatdata relating to the present disclosure can be transmitted over suchnetworks or connections for reception and/or review by a party 1922 asillustrated in FIG. 19.

FIG. 20 is a block diagram illustrating a first example architecture ofa computer system 2000 that can be used in connection with exampleembodiments of the present invention. As depicted in FIG. 20, theexample computer system can include a processor 2002 for processinginstructions. Non-limiting examples of processors include: Intel Xeon™processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-Sv1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8Apple A4™ processor, Marvell PXA 930™ processor, or afunctionally-equivalent processor. Multiple threads of execution can beused for parallel processing. In some embodiments, multiple processorsor processors with multiple cores can also be used, whether in a singlecomputer system, in a cluster, or distributed across systems over anetwork comprising a plurality of computers, cell phones, and/orpersonal data assistant devices.

As illustrated in FIG. 20, a high speed cache 2004 can be connected to,or incorporated in, the processor 2002 to provide a high speed memoryfor instructions or data that have been recently, or are frequently,used by processor 2002. The processor 2002 is connected to a northbridge 2006 by a processor bus 2008. The north bridge 2006 is connectedto random access memory (RAM) 2010 by a memory bus 2012 and managesaccess to the RAM 2010 by the processor 2002. The north bridge 2006 isalso connected to a south bridge 2014 by a chipset bus 2016. The southbridge 2014 is, in turn, connected to a peripheral bus 2018. Theperipheral bus can be, for example, PCI, PCI-X, PCI Express, or otherperipheral bus. The north bridge and south bridge are often referred toas a processor chipset and manage data transfer between the processor,RAM, and peripheral components on the peripheral bus 2018. In somealternative architectures, the functionality of the north bridge can beincorporated into the processor instead of using a separate north bridgechip.

In some embodiments, system 2000 can include an accelerator card 2022attached to the peripheral bus 2018. The accelerator can include fieldprogrammable gate arrays (FPGAs) or other hardware for acceleratingcertain processing. For example, an accelerator can be used for adaptivedata restructuring or to evaluate algebraic expressions used in extendedset processing.

Software and data are stored in external storage 2024 and can be loadedinto RAM 2010 and/or cache 2004 for use by the processor. The system2000 includes an operating system for managing system resources;non-limiting examples of operating systems include: Linux, Windows™,MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalentoperating systems, as well as application software running on top of theoperating system for managing data storage and optimization inaccordance with example embodiments of the present invention.

In this example, system 2000 also includes network interface cards(NICs) 2020 and 2021 connected to the peripheral bus for providingnetwork interfaces to external storage, such as Network Attached Storage(NAS) and other computer systems that can be used for distributedparallel processing.

FIG. 21 is a diagram showing a network 2100 with a plurality of computersystems 2102 a, and 2102 b, a plurality of cell phones and personal dataassistants 2102 c, and Network Attached Storage (NAS) 2104 a, and 2104b. In example embodiments, systems 2102 a, 2102 b, and 2102 c can managedata storage and optimize data access for data stored in NetworkAttached Storage (NAS) 2104 a and 2104 b. A mathematical model can beused for the data and be evaluated using distributed parallel processingacross computer systems 2102 a, and 2102 b, and cell phone and personaldata assistant systems 2102 c. Computer systems 2102 a, and 2102 b, andcell phone and personal data assistant systems 2102 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 2104 a and 2104 b. FIG. 21 illustratesan example only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various embodiments of thepresent invention. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface.

In some example embodiments, processors can maintain separate memoryspaces and transmit data through network interfaces, back plane or otherconnectors for parallel processing by other processors. In otherembodiments, some or all of the processors can use a shared virtualaddress memory space.

FIG. 22 is a block diagram of a multiprocessor computer system 2200using a shared virtual address memory space in accordance with anexample embodiment. The system includes a plurality of processors 2202a-f that can access a shared memory subsystem 2204. The systemincorporates a plurality of programmable hardware memory algorithmprocessors (MAPs) 2206 a-f in the memory subsystem 2204. Each MAP 2206a-f can comprise a memory 2208 a-f and one or more field programmablegate arrays (FPGAs) 2210 a-f. The MAP provides a configurable functionalunit and particular algorithms or portions of algorithms can be providedto the FPGAs 2210 a-f for processing in close coordination with arespective processor. For example, the MAPs can be used to evaluatealgebraic expressions regarding the data model and to perform adaptivedata restructuring in example embodiments. In this example, each MAP isglobally accessible by all of the processors for these purposes. In oneconfiguration, each MAP can use Direct Memory Access (DMA) to access anassociated memory 2208 a-f, allowing it to execute tasks independentlyof, and asynchronously from, the respective microprocessor 2202 a-f. Inthis configuration, a MAP can feed results directly to another MAP forpipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleembodiments, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some embodiments,all or part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example embodiments, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

In example embodiments, the computer system can be implemented usingsoftware modules executing on any of the above or other computerarchitectures and systems. In other embodiments, the functions of thesystem can be implemented partially or completely in firmware,programmable logic devices such as field programmable gate arrays(FPGAs) as referenced in FIG. 22, system on chips (SOCs), applicationspecific integrated circuits (ASICs), or other processing and logicelements. For example, the Set Processor and Optimizer can beimplemented with hardware acceleration through the use of a hardwareaccelerator card, such as accelerator card 122 illustrated in FIG. 20.

Example 1: Front-End Processing of a Silicon Wafer to Create a Microwell

Silicon wafers are etched to create an exemplary substrate comprising aplurality of microwells using a front-end processing method asillustrated in FIG. 23. Starting with a SOI substrate with a layer ofoxide on both surfaces of the substrate, a layer of photo-resist iscoated using photolithography method on the handle-side of the substrateat preferred locations. Following the coating of the photo-resist, DRIEis performed on the handle side until reaching to the layer of oxide inthe middle of the wafer. Then, the coating of the photo-resist isstripped away exposing the layer of oxide underneath. Similarly, asecond layer of photo-resist is coated using photolithography method onthe device-side of the substrate at preferred locations, with suitablediameters. Following the coating of the second layer of photo-resist,DRIE is performed again on the device-side of the silicon wafer untilreaching the layer of oxide in the middle of the silicon wafer. Then,the photo-resist and the layer of oxide in the middle of the wafer isstripped away. Lastly, oxide is coated on all surface of the wafer,creating a silicon wafer with a plurality of microstructures, eachcomprising a larger microwell and one or more microchannels fluidicallyconnected to the microwell.

Example 2: Back-End Processing of a Silicon Wafer to FunctionalizeSelected Surface of the Microwell

The silicon wafer with etched microwells is further processed tofunctionalize selected portions of the microwells using a back-endprocessing method as illustrated in FIG. 24. To coat only the surface ofa smaller microwell within a microwell with an active functionalizationagent that increases surface energy, the product from Example 1 is usedas the starting material. A droplet of photo-resist is deposited intothe microchannel using an inkjet printer as described herein. Thedroplet of photo-resist is spread into the microchannel in fluidicconnection to the microwell. Following the photoresist deposition,oxygen plasma etch is performed to etch back excess photoresist, leavinga smoother surface of photo-resist as illustrated in FIG. 24. A layer ofa chemically inert moiety is coated onto all exposed surfaces of thesilicon wafer to create a passive functionalization layer with lowsurface energy. Afterwards, the photo-resist is stripped away, exposingthe surface of the smaller microchannel in fluidic communication withthe microwell. Upon removal of the photo-resist, a layer of activefunctionalization agent is coated onto the surface of the smallermicrochannel to increase the surface energy of the surface of themicrowell and/or to provide surface chemistries for oligonucleotidegrowth. The previously functionalized surfaces remain substantiallyunaffected by the second application of surface functionalization. As aresult, a plurality of microwell with a first surface functionalizationeach in fluidic communication with one or more microchannels with asecond surface functionalization is manufactured on a solid substrate.

Example 3: Microfluidic Device

A microfluidic device comprising a substantially planar substrateportion was manufactured according to the methods and compositions ofthe invention as shown in FIG. 25D. A cross-section of the substrate isshown in FIG. 25E. The substrate comprises 108 clusters, wherein eachcluster comprises 109 groupings of fluidic connections. Each groupingcomprises 5 second channels extending from a first channel. FIG. 25A isa device view of each cluster comprising the 109 groupings. FIG. 25C isa handle view of the cluster of FIG. 25 part 25A. FIG. 25B is a sectionview of FIG. 25A showing a row of 11 groupings. FIG. 25F is another viewof the substrate shown in FIG. 25D, wherein the position of a label isvisualized. FIG. 25G is an expanded view of FIG. 25A, indicating the 109groupings of the cluster.

As shown in FIGS. 25A and 25C, the 109 groupings are arranged in offsetrows to form a cluster in a circle-like pattern, where the individualregions are non-overlapping with each other. The individual groupingsform a circle. As represented by 2503, the distance between three rowsof these groupings is 0.254 mm. As shown by 2506, the distance betweentwo groupings in a row of groupings is 0.0978 mm. The cross-section ofthe first channel in a grouping, as shown by 2504, is 0.075 mm. Thecross-section of each second channel in a grouping, as shown by 2505, is0.020 mm. The length of the first channel in a grouping, as shown by2502, is 0.400 mm. The length of each second channel in a grouping, asshown by 2501, is 0.030 mm.

The cluster of 109 groupings shown in FIGS. 25A and 25C are arranged ina conformation suitable for placement in a single reaction well that maybe placed adjacent to the cluster in FIGS. 25A and 25C. The remainder ofthe clusters in FIG. 25D are similarly arranged in a way thatfacilitates delivery into a number of reaction wells, such as thenanoreactor plate described in FIGS. 26A-26E and Example 4. Thesubstrate comprises 108 reaction wells, providing 11,772 groupings.

The width of the substrate along one dimension, as indicated by 2508, is32.000 mm. The width of the substrate along another dimension, asindicated by 2519, is 32.000 mm.

The substantially planar substrate portion, as shown in FIG. 25D,comprises 108 clusters of groupings. The clusters are arranged in rowsforming a square shape. The furthest distance from the center of acluster to the origin in one dimension, as indicated by 2518, is 24.467mm. The furthest distance from the center of a cluster to the origin inanother dimension, as indicated by 2509, is 23.620 mm. The closestdistance from the center of a cluster to the origin in one dimension, asshown by 2517, is 7.533. The closest distance from the center of acluster to the origin in another dimension, as shown by 2512, is 8.380.The distance between the centers of two clusters in the same row, asshown by 2507 and 2522 is 1.69334 mm.

The substrate comprises 3 fiducial marks to facilitate alignment of themicrofluidic device with other components of a system. A first fiducialmark is located near the origin, where the fiducial mark is closer tothe origin than any one cluster. The first fiducial mark is located5.840 mm from the origin in one dimension (2516) and 6.687 mm from theorigin in another dimension (2513). The first fiducial mark is located1.69334 mm from a cluster in one dimension (2515) and 1.69344 mm fromthe same cluster in another dimension (2514). Two other fiducial marksare each located 0.500 mm from an edge of the substrate (2510 and 2520)and 16.000 mm (2511 and 2521) from the origin.

A cross section of the substrate is shown in FIG. 25E, where the totallength of a grouping as indicated by 2523, is 0.430 mm.

Another view of the substrate is shown is shown in FIG. 25F, showing thearrangement of the 108 clusters and the position of a label. The labelis located 1.5 mm (2603) from an edge of the substrate. The label islocated at a distance between 4.0 mm (2602) to 9.0 mm (2601), asmeasured from the origin.

Example 4: Nanoreactor

An nanoreactor was manufactured according to the methods andcompositions of the invention as shown in FIGS. 26B and 26C. Across-section of the nanoreactor is shown in FIG. 26A. The nanoreactorcomprises 108 wells. FIG. 26D is a handle view of a nanoreactor. FIG.26E is another view of the nanoreactor shown in FIG. 26B, wherein theposition of a label is visualized.

As shown in FIG. 26B, the 108 wells are arranged in rows to form asquare pattern, where the individual wells are raised on the nanoreactorbase. As shown by 2711, the distance between the centers of two wells ina row of wells is 1.69334 mm. The cross-section of the inside of a well,as shown by 2721, is 1.15 mm. The cross-section of a well, including therim of the well, as shown by 2720, is 1.450 mm. The height of a well ina nanoreactor, as shown by 2702, is 0.450 mm. The total height of ananoreactor, as shown by 2701, is 0.725 mm

The wells in FIG. 26B are arranged in a way that facilitates deliveryfrom a microfluidic device having 108 wells, as exemplified by FIG. 26,into the 108 reaction wells of the nanoreactor.

The width of the nanoreactor along one dimension, as indicated by 2703,is 24.000 mm. The width of the nanoreactor along another dimension, asindicated by 2704, is 24.000 mm.

The nanoreactor, as shown in FIG. 26B, comprises 108 wells. The wellsare arranged in rows forming a square shape. The furthest distance fromthe center of a well to the origin in one dimension, as indicated by2706, is 20.467 mm. The furthest distance from the center of a well tothe origin in another dimension, as indicated by 2705, is 19.620 mm. Theclosest distance from the center of a well to the origin in onedimension, as shown by 2710, is 3.533 mm. The closest distance from thecenter of a well to the origin in another dimension, as shown by 2709,is 4.380 mm. The distance between the centers of two wells in the samerow, as shown by 2711 and 2712 is 1.69334 mm. The distance from thecenter of a well to the edge of a nanoreactor in one dimension, as shownby 2707, is 3.387 mm. The distance from the center of a well to the edgeof a nanoreactor in another dimension, as shown by 2708, is 2.540 mm.

The nanoreactor comprises 3 fiducial marks on the device face tofacilitate alignment of the nanoreactor with other components of asystem, for example, a microfluidic device as described in Example 3. Afirst fiducial mark is located near the origin, where the fiducial markis closer to the origin than any one well. The first fiducial mark islocated 1.840 mm from the origin in one dimension (2717) and 2.687 mmfrom the origin in another dimension (2716). The first fiducial mark islocated 1.6933 mm from a well in one dimension (2719) and 1.6934 mm fromthe same well in another dimension (2718). Two other fiducial marks areeach located 0.500 mm from an edge of the nanoreactor (2714 and 2715)and 12.000 mm (2713) from the origin.

The nanoreactor comprises 4 fiducial marks on the handle face as shownin FIG. 26D. The distance from the center or a fiducial mark and anearest corner of the nanoreactor in one dimension is 1.000 mm (2722 and2723). The length of a fiducial mark in one dimension is 1.000 mm (2724and 2725). The width of a fiducial mark, as shown by 2726, is 0.050 mm.

Another view of the nanoreactor is shown is shown in FIG. 26E, showingthe arrangement of the 108 wells and the position of a label. The labelis located 1.5 mm (2728) from an edge of the nanoreactor. The label islocated 1.0 mm (2727) from a corner of the nanoreactor. The label is 9.0mm (2726), in length.

Example 5: Manufacturing of an Oligonucleotide Synthesis Device

A silicon on insulator (SOI) wafer with an about 30 um thick devicelayer and an about 400 um thick handle layer sandwiching an electricalinsulator layer of silicon dioxide was etched to create the exemplarysubstrate described in Example 3 comprising a plurality of featureshaving three-dimensional microfluidic connections, using a front-endprocessing method as illustrated in FIG. 28. FIG. 27 illustrates indetail the design features of the device. The SOI wafer was oxidized tocover it with thermal oxide on both surfaces (FIG. 28 part A).Photolithography was applied to the device side to create a mask ofphotoresist (red) as shown in FIG. 28 part B. A deep reactive-ionetching (DRIE) step was used to etch vertical side-walls to a depth ofabout 30 um up until the SOI oxide layer (FIG. 28 part C) at locationsdevoid of the photoresist. The photoresist was stripped using standardresist stripping process known in the art.

The photolithography, DRIE, and stripping of photoresist was repeated onthe handle side (FIG. 28 part E to part G) to generate the desiredpattern according to the device described in Example 3. The buried oxide(BOX) was removed using a wet etch process (FIG. 28 part G).Contaminating fluoropolymers that may have been deposited on the sidewalls of the microfluidic features were removed by thermal oxidation.The thermal oxidation was stripped using a wet etching process.

The etched SOI wafers were subjected to processing steps as described inFIG. 29 parts A-F.

First, the wafer was cleaned by a wet cleaning step using piranhasolution followed by a dry O₂ plasma exposure. The device layer (on topin FIG. 29 part B) of the chip was coated with photoresist in a processgoverned by wicking into the device layer channels that are about 20 umwide. The photoresist was patterned using photolithography to expose theareas that are desired to be passive (no future oligonucleotidesynthesis). This process works by exposing the resist to light through abinary mask that has the pattern of interest. After exposure, the resistin the exposed regions was removed in developer solution. (FIG. 29 partC).

The surfaces without photoresist were exposed to a fluorosilane gas bychemical vapor deposition (CVD). This results in the deposition of afluorocarbon on the surfaces without photoresist. In alternativeapplications, a hydrocarbon silane is used for this step. The silanizedsurfaces are unresponsive to additional layers of silane creating amonolayer on the surface. The photoresist was then dissolved in organicsolvent, leaving fluorination on the surface and exposingsilicon/silicon dioxide that was underneath the photoresist. A finalstep of active functionalization was performed to prepare the surfacefor oligonucleotide growth (FIG. 29 part F).

A controlled surface density of hydroxyl groups (FIG. 30) was achievedon the surface by a wet process using a 1% solution ofN-(3-TRIETHOXYSILYLPROPYL-4HYDROXYBUTYRAMIDE in ethanol and acetic acidfor 4 hours, followed by putting the chips on a hot plate at 150 C for14 hours. In alternative applications, a CVD process is performed bydelivering silane to the surface in gaseous state and applying acontrolled deposition pressure of about 200 mTor and a controlledtemperature of about 150 C. The CVD process allows for in-situ plasmacleaning and is well suited for producing highly ordered self-assembledmonolayers (SAMs).

FIGS. 31A-31B shows an image of a device manufactured according to themethods above.

Example 6: Manufacturing of a Nanoreactor Device

A nanoreactor chip with nanowells as described in FIG. 32 wasmanufactured. A suitable sized silicon wafer was oxidized to cover itwith thermal oxide on both surfaces (FIG. 33 part A).

Photolithography was applied to the back side to create a mask ofphotoresist (red) as shown in FIG. 33 part B. The back side was etchedat locations devoid of the photoresist, beyond the thermal oxide layer,creating shallow wells (FIG. 33 part C). The photoresist was strippedusing standard resist stripping process known in the art (FIG. 33 partD).

The photolithography step was repeated on the front side according tothe pattern in FIG. 33 part E. A deep reactive-ion etching (DRIE) stepwas used to etch vertical side-walls to a depth of about 450 um using atimed etch. In other cases, a SOI wafer is used and the handle layer isetched down to the BOX, wherein the BOX can serve as an etch stop. (FIG.33 part F). The photoresist on the front side was stripped (FIG. 33 partG), generating the desired pattern according to the device described inFIG. 32. Contaminating fluoropolymers that may have been deposited onthe side walls of the microfluidic features were removed by thermaloxidation and the thermal oxidation was stripped using a wet etchingprocess (FIG. 33 part H).

Next, the wafer was cleaned by a wet cleaning step using piranhasolution followed by a dry O₂ plasma exposure (FIG. 34 part A). Resistwas then deposited into individual wells using a microdrop depositionsystem (top, in FIG. 34 part B). The surfaces without resist wereexposed to a fluorosilane gas by chemical vapor deposition (CVD; FIG. 34part C). This results in the deposition of a fluorocarbon on thesurfaces without the resist. In alternative applications, a hydrocarbonsilane or other types of silanes are used for this step. The silanizedsurfaces are unresponsive to additional layers of silane creating amonolayer on the surface. The resist was then dissolved in organicsolvent, leaving fluorination on the surface and exposing the siliconsurface that was underneath the resist.

FIG. 35 parts A-B illustrate the nanowells in a nanoreactor devicemanufactured as described.

Example 7: Synthesis of a 50-mer Sequence on a 2D OligonucleotideSynthesis Device

A two dimensional oligonucleotide synthesis device was assembled into aflowcell, which was connected to an flowcell (Applied Biosystems (ABI394DNA Synthesizer”). The two-dimensional oligonucleotide synthesis devicewas uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest,shop.gelest.com/Product.aspx?catnum=SIT8189.5&Index=0&TotalCount=1) wasused to synthesize an exemplary oligonucleotide of 50 bp (“50-meroligonucleotide”) using oligonucleotide synthesis methods describedherein.

The sequence of the 50-mer was as described in SEQ ID NO.: 1.

5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCA T##TTTTTTTTTT3′ (SEQID NO.: 1), where # denotes Thymidine-succinyl hexamide CEDphosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linkerenabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 3and an ABI synthesizer. Table 3:

TABLE 3 General DNA Synthesis Process Name Process Step Time (sec) WASH(Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile toFlowcell 23 N2 System Flush 4 Acetonitrile System Flush 4 DNA BASEADDITION Activator Manifold Flush 2 (Phosphoramidite + Activator toFlowcell 6 Activator Flow) Activator + 6 Phosphoramidite to FlowcellActivator to Flowcell 0.5 Activator + 5 Phosphoramidite to FlowcellActivator to Flowcell 0.5 Activator + 5 Phosphoramidite to FlowcellActivator to Flowcell 0.5 Activator + 5 Phosphoramidite to FlowcellIncubate for 25 sec 25 WASH (Acetonitrile Wash Acetonitrile System Flush4 Flow) Acetonitrile to Flowcell 15 N2 System Flush 4 AcetonitrileSystem Flush 4 DNA BASE ADDITION Activator Manifold Flush 2(Phosphoramidite + Activator to Flowcell 5 Activator Flow) Activator +18 Phosphoramidite to Flowcell Incubate for 25 sec 25 WASH (AcetonitrileWash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2System Flush 4 Acetonitrile System Flush 4 CAPPING (CapA + B, 1:1,CapA + B to Flowcell 15 Flow) WASH (Acetonitrile Wash AcetonitrileSystem Flush 4 Flow) Acetonitrile to Flowcell 15 Acetonitrile SystemFlush 4 OXIDATION (Oxidizer Oxidizer to Flowcell 18 Flow) WASH(Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2 System Flush 4Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 AcetonitrileSystem Flush 4 Acetonitrile to Flowcell 15 N2 System Flush 4Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 System Flush4 Acetonitrile System Flush 4 DEBLOCKING (Deblock Deblock to Flowcell 36Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 18N2 System Flush 4.13 Acetonitrile System Flush 4.13 Acetonitrile toFlowcell 15

The phosphoramidite/activator combination was delivered similar to thedelivery of bulk reagents through the flowcell. No drying steps wereperformed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enablefaster flow. Without flow restrictor, flow rates for amidites (0.1M inACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx fromGlenResearch) in ACN), and Ox (0.02M I2 in 20% pyridine, 10% water, and70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and cappingreagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride inTHF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flowrestrictor).

The time to completely push out Oxidizer was observed, the timing forchemical flow times was adjusted accordingly and an extra ACN wash wasintroduced between different chemicals.

After oligonucleotide synthesis, the chip was deprotected in gaseousammonia overnight at 75 psi. Five drops of water were applied to thesurface to recover oligos (FIG. 45 part A). The recovered oligos werethen analyzed on a BioAnalyzer small RNA chip (FIG. 45 part B).

Example 8: Synthesis of a 100-mer Sequence on a 2D OligonucleotideSynthesis Device

The same process as described in Example 7 for the synthesis of the50-mer sequence was used for the synthesis of a 100-mer oligonucleotide(“100-mer oligonucleotide”; 5′CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3′, where #denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 fromChemGenes); SEQ ID NO.: 2) on two different silicon chips, the first oneuniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second onefunctionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane andn-decyltriethoxysilane, and the oligos extracted from the surface wereanalyzed on a BioAnalyzer instrument (FIG. 46).

All ten samples from the two chips were further PCR amplified using aforward (5′ATGCGGGGTTCTCATCATC3′; SEQ ID NO.: 3) and a reverse(5′CGGGATCCTTATCGTCATCG3′; SEQ ID NO.: 4) primer in a 50 uL PCR mix (25uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverseprimer, 1 uL oligo extracted from the surface, and water up to 50 uL)using the following thermalcycling program:

98 C, 30 sec

98 C, 10 sec; 63 C, 10 sec; 72 C, 10 sec; repeat 12 cycles

72 C, 2 min

The PCR products were also run on a BioAnalyzer (FIG. 47), demonstratingsharp peaks at the 100-mer position.

Next, the PCR amplified samples were cloned, and Sanger sequenced. Table4 summarizes the results from the Sanger sequencing for samples takenfrom spots 1-5 from chip 1 and for samples taken from spots 6-10 fromchip 2.

TABLE 4 Spot Error rate Cycle efficiency 1 1/763 bp 99.87% 2 1/824 bp99.88% 3 1/780 bp 99.87% 4 1/429 bp 99.77% 5 1/1525 bp 99.93% 6 1/1615bp 99.94% 7 1/531 bp 99.81% 8 1/1769 bp 99.94% 9 1/854 bp 99.88% 101/1451 bp 99.93%

Thus, the high quality and uniformity of the synthesizedoligonucleotides were repeated on two chips with different surfacechemistries. Overall, 89%, corresponding to 233 out of 262 of the100-mers that were sequenced were perfect sequences with no errors.

FIGS. 48 and 49 show alignment maps for samples taken from spots 8 and7, respectively, where “×” denotes a single base deletion, “star”denotes single base mutation, and “+” denotes low quality spots inSanger sequencing. The aligned sequences in FIG. 48 together representan error rate of about 97%, where 28 out of 29 reads correspond toperfect sequences. The aligned sequences in FIG. 49 together representan error rate of about 81%, where 22 out of 27 reads correspond toperfect sequences.

Finally, Table 5 summarizes key error characteristics for the sequencesobtained from the oligonucleotides samples from spots 1-10.

TABLE 5 Sample ID/ Spot no. OSA_0046/1 OSA_0047/2 OSA_0048/3 OSA_0049/4OSA_0050/5 Total Sequences 32 32 32 32 32 Sequencing 25 of 28 27 of 2726 of 30 21 of 23 25 of 26 Quality Oligo Quality 23 of 25 25 of 27 22 of26 18 of 21 24 of 25 ROI Match Count 2500 2698 2561 2122 2499 ROIMutation 2 2 1 3 1 ROI MultiBase 0 0 0 0 0 Deletion ROI Small 1 0 0 0 0Insertion ROI Single Base 0 0 0 0 0 Deletion Large Deletion 0 0 1 0 0Count Mutation: G > A 2 2 1 2 1 Mutation: T > C 0 0 0 1 0 ROI ErrorCount 3 2 2 3 1 ROI Error Rate Err: ~1 in Err: ~1 in Err: ~1 in Err: ~1in Err: ~1 in 834 1350 1282 708 2500 ROI Minus Primer MP Err: ~1 MP Err:~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 Error Rate in 763 in 824 in 780 in429 in 1525 Sample ID/ Spot no. OSA_0051/6 OSA_0052/7 OSA_0053/8OSA_0054/9 OSA_0055/10 Total Sequences 32 32 32 32 32 Sequencing 29 of30 27 of 31 29 of 31 28 of 29 25 of 28 Quality Oligo Quality 25 of 29 22of 27 28 of 29 26 of 28 20 of 25 ROI Match Count 2666 2625 2899 27982348 ROI Mutation 0 2 1 2 1 ROI MultiBase 0 0 0 0 0 Deletion ROI Small 00 0 0 0 Insertion ROI Single Base 0 0 0 0 0 Deletion Large Deletion 1 10 0 0 Count Mutation: G > A 0 2 1 2 1 Mutation: T > C 0 0 0 0 0 ROIError Count 1 3 1 2 1 ROI Error Rate Err: ~1 in Err: ~1 in Err: ~1 inErr: ~1 in Err: ~1 in 2667 876 2900 1400 2349 ROI Minus Primer MP Err:~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 MP Err: ~1 Error Rate in 1615 in 531in 1769 in 854 in 1451

Example 9: Synthesis of a 100-mer Sequence on a 3D OligonucleotideSynthesis Device

A three dimensional oligonucleotide synthesis device as described inExample 3 that was differentially functionalized with a 5/95 mix of11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane on activeareas for synthesis was assembled into a flowcell to synthesize the100-mer oligonucleotide of Example 8 using oligonucleotide synthesismethods described herein. The synthesis was done using standard DNAsynthesis chemistry (coupling, capping, oxidation, and deblocking) asdescribed in Example 7, according to the protocol in Table 3. The chipwas deprotected in gaseous ammonia, at 75 psi, overnight and the oligoswere eluted in 500 uL water. After evaporation, all oligos werere-suspended in 20 uL water for downstream analysis. The re-suspendedsample was analyzed on a BioAnalzyer instrument (FIG. 50 part A).

The re-suspended sample was also PCR amplified using forward(5′ATGCGGGGTTCTCATCATC3′; SEQ ID NO.: 5) and reverse(5′CGGGATCCTTATCGTCATCG3′; SEQ ID NO.: 6) primers in a 50 uL PCR mixincluding 25 uL NEB Q5 mastermix, 2.5 uL 10 uM forward primer, 2.5 uL 10uM reverse primer, 1 uL oligo extracted from the surface, and water upto 50 uL, according to the following thermalcycling program:

1 cycle: 98 C, 30 sec

12 cycles: 98 C, 10 sec; 63 C, 10 sec; 72 C, 10 sec

1 cycle: 72 C, 2 min

The PCR product was also run on the BioAnalyzer (FIG. 50 part B) showinga sharp peak at the 100-mer position.

The sequencing result of the PCR products showed that 23 out of 29sequences were perfect and error rate was ˜1 in 600 bp as illustrated bythe alignment maps in FIG. 51, where “×” denotes a single base deletion,“star” denotes single base mutation, and “+” denotes low quality spotsin Sanger sequencing.

Example 10: Parallel Oligonucleotide Synthesis on a Three DimensionalMicrofluidic Oligonucleotide Synthesis Device

The synthesis protocol of EXAMPLE 7 is modified using a house set-up toperform parallel oligonucleotide synthesis on the three dimensionalmicrofluidic device of EXAMPLE 9.

Table 6 illustrates a side by side comparison of the two protocols.

TABLE 6 Twist In-House Synthesizer General DNA EXAMPLE 7 ProtocolProtocol Synthesis Process Time Time Name EXAMPLE 7 Process Step (sec)Twist Process Step (sec) WASH (Acetonitrile Acetonitrile System Flush 4NA Wash Flow) Acetonitrile to Flowcell 23 N2 System Flush 4 AcetonitrileSystem Flush 4 DNA BASE Activator Manifold Flush 2 Print heads print 1:1of 120 ADDITION Activator to Flowcell 6 Activator + (Phosphoramidite +Activator + Phosphoramidite 6 Phosphoramidite directly Activator Flow)to Flowcell on chip active sites Activator to Flowcell 0.5 Activator +Phosphoramidite 5 to Flowcell Activator to Flowcell 0.5 Activator +Phosphoramidite 5 to Flowcell Activator to Flowcell 0.5 Activator +Phosphoramidite 5 to Flowcell Incubate for 25 sec 25 WASH (AcetonitrileAcetonitrile System Flush 4 Wash Flow) Acetonitrile to Flowcell 15 N2System Flush 4 Acetonitrile System Flush 4 DNA BASE Activator ManifoldFlush 2 ADDITION Activator to Flowcell 5 (Phosphoramidite + Activator +Phosphoramidite 18 Activator Flow) to Flowcell Incubate for 25 sec 25WASH (Acetonitrile Acetonitrile System Flush 4 Acetonitrile System Flush4 Wash Flow) Acetonitrile to Flowcell 15 Acetonitrile to Flowcell 15 N2System Flush 4 N2 System Flush 4 Acetonitrile System Flush 4Acetonitrile System Flush 4 CAPPING (CapA + B, CapA + B to Flowcell 15CapA + B to Flowcell 15 1:1, Flow) WASH (Acetonitrile AcetonitrileSystem Flush 4 Acetonitrile System Flush 4 Wash Flow) Acetonitrile toFlowcell 15 Acetonitrile to Flowcell 15 Acetonitrile System Flush 4Acetonitrile System Flush 4 OXIDATION Oxidizer to Flowcell 18 Oxidizerto Flowcell 18 (Oxidizer Flow) WASH (Acetonitrile Acetonitrile SystemFlush 4 Acetonitrile System Flush 4 Wash Flow) N2 System Flush 4 N2System Flush 4 Acetonitrile System Flush 4 Acetonitrile System Flush 4Acetonitrile to Flowcell 15 Acetonitrile to Flowcell 15 AcetonitrileSystem Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15Acetonitrile to Flowcell 15 N2 System Flush 4 N2 System Flush 4Acetonitrile System Flush 4 Acetonitrile System Flush 4 Acetonitrile toFlowcell 23 Acetonitrile to Flowcell 23 N2 System Flush 4 N2 SystemFlush 4 Acetonitrile System Flush 4 Acetonitrile System Flush 4DEBLOCKING Deblock to Flowcell 36 Deblock to Flowcell 36 (Deblock Flow)WASH (Acetonitrile Acetonitrile System Flush 4 Acetonitrile System Flush4 Wash Flow) N2 System Flush 4 N2 System Flush 4 Acetonitrile SystemFlush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 18Acetonitrile to Flowcell 18 N2 System Flush 4 N2 System Flush 4Acetonitrile System Flush 4 Acetonitrile System Flush 4 Acetonitrile toFlowcell 15 Acetonitrile to Flowcell 15 FLOWCELL DRY NA N2 System Flush4 (Specific to Twist N2 to Flowcell 19.5 synthesizer) N2 System Flush 4Vacuum Dry Pull on 10 Flowcell N2 System Flush 4 N2 to Flowcell 19.5

Acetonitrile (ACN) is passed through an in-line degasser (Model No.403-0202-1; Random Technologies), passing the liquid along side a veryhydrophobic membrane, which was previously shown to function at flowrates ranging from 50-400 uL/sec and to eliminate gas bubbles that formon a flow cell, without being bound by theory, likely by dissolving themin the undersaturated solvent.

Reagents are exchanged in the flowcell with different reagents asfollows:

-   -   1) Start reagent flow to the flowcell.    -   2) Prime by setting the valves to “push” the previous reagent        out of the delivery line with the new reagent. This valve state        is kept on for 3.75 sec.    -   3) 2D Valve State: Set the valves to replace the previous        reagent resident on the surface of the flowcell with the new        reagent. This occurs whilst step 2 has been active for 3.75 sec.        Step 2 and 3 are simultaneously active for 0.25 sec, after which        the priming valve state turns off.    -   4) 3D Valve State: The valves switch to allow for reagents to        flow through the three-dimensional microfluidic features of the        silicon in the flowcell, which starts after 0.75 sec of the 2D        Valve State in step 3 has flowed.    -   5) The flow of reagent: 2D valve state and 3D valve states        remain open for a designated time to allow for adequate dosage        of reagent to the silicon surface in the chip.        Accordingly, during a 5 second cycle of reagent exchange, the        fluid delivery is performed by priming during the initial period        spanning 0-4 seconds, by turning on the 2D Valve State during        the period spanning the 3.75-5 seconds and by turning on the 3D        Valve State during the period spanning 4.5-5 seconds.

The phosphoramidite/activator combination is delivered using an ink jetprinting step. The delivery can be a 1:1, drop-on-drop deposition ontothe silicon surface. The droplet size may be about 10 pL. In someembodiments, the droplet size is at least or at least about 0.1, 1, 2,3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500picoliters, or more. In some embodiments, the droplet size is at most orat most about 500, 400, 300, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10,5, 4, 3, 2, 1, 0.1 picoliters, or less. The droplet size may be between0.1-50, 1-150, or 5-75 picoliters. The droplet size may fall within arange that is bound by any of these values, e.g. 2-50 picoliters. Thedroplets may be deposited with an initial velocity of 1-100 m/sec. Insome cases, the droplets may be deposited with an initial velocity of atleast or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40,35, 40, 45, 50, 75, 100 m/sec, or higher. In some cases, the dropletsmay be deposited with an initial velocity of at most or at most about100, 75, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1,m/sec or lower. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 40, 35, 40,45, 50, 75, 100, or higher. The droplets may be deposited with aninitial velocity that falls between about 1-50 m/sec., 5-15 m/sec, 5-30m/sec, or 1-30 m/sec. Those having skill in the art will understand thatthe droplets may be deposited with an initial velocity that falls undera range bound by any of these values.

A drying step prepares the silicon surface for the printing steps afterthe bulk reagent sequences. To achieve dry conditions which facilitateprinted reagents to react, the flowcell is flushed with N₂ gas at about5 PSI for about 19.5 seconds, a small vacuum is pulled on the flowcellchamber for 10 seconds, and the flowcell is flushed again with N2 gasfor another 19.5 seconds. All reagents are flown at about 200-400 uL/sec

Flow rates can be controlled in the in-house system using varyingpressures. Flow rate is one of the limiting aspects of commerciallyavailable synthesizers. In the in-house machine set-up, flow rates caneither be matched to their values in Example 7 or to increased ordecreased flowrates, as appropriate, to improve the synthesis process.Generally, flowing faster presents advantages as it allows to displacebubbles much more effectively and allows for more exchange of freshreagents to the surface during a given time interval when compared withthe slower flow rates.

Example 11: Blotting Based Oligonucleotide Transfer from anOligonucleotide Synthesis Device to a Nanoreactor Device

50-mer oligonucleotides were synthesized on a 3-D oligonucleotidesynthesis device as described in Example 9. No active functionalizationwas applied. FIG. 53 parts A-B illustrates the oligonucleotide synthesischannel distribution in a cluster on the device side of theoligonucleotide synthesis device and FIG. 53 part C illustrates thesurface functionalization. The oligonucleotides were released from thesurface by treatment in a gaseous ammonia chamber at 75 psi for 14hours.

Wells of a nanoreactor device that was manufactured according to Example4 with hydrophilic inner walls and hydrophobic top lips (FIG. 54) werefirst filled with a PCA suitable buffer as a negative control (5× Q5buffer; New England Biolabs). 200-300 nL aliquots were hand-pipetted tofeed into a BioAnalyzer to show the absence of any contaminating nucleicacids in the individual nanoreactors (FIG. 55).

The nanoreactors were next filled with about 650 nL of PCA bufferforming a meniscus that slightly bulged out (FIG. 53). The nanoreactordevice was mated with the oligonucleotide device to submerge theoligonucleotide synthesis channels (“revolver”) with the PCA buffer at arate of about 5 mm/sec. In other cases, the mantling velocity for matingthe two devices may be varied as described herein, to achieve, amongother things, more or less efficient liquid transfer between the devicesgiving rise to controlled aliquoting of desired volumes of liquid or tocontrol evaporation. The oligonucleotide device and the nanoreactor werekept mated with a gap of about 50 um between the two devices, for about10 minutes, allowing the oligonucleotides to diffuse into the solution(FIG. 57). In some cases, the assembly or the oligonucleotide synthesisdevice alone can be vibrated or oscillated to facilitate fasterdiffusion. Diffusion times longer than 10 min, such as at least or atleast about 11, 12, 13, 14, 15, 20, 25 min, or longer may also be usedto facilitate higher yield. The nanoreactor device was released from theoligonucleotide device at a rate of about 5 mm/sec, capturing thereleased oligonucleotides in the individual nanoreactors. In othercases, the dismantling velocity for mating the two devices may be variedas described herein, to achieve, among other things, more or lessefficient liquid transfer between the devices giving rise to controlledaliquoting of desired volumes of liquid. A tiny amount of liquid wasobserved to be left over on the oligonucleotide device.

Samples of about 300 nL were pipetted out from several individualnanoreactors in the nanoreactor device and diluted into a volume of 1uL, establishing a 4.3× dilution. The diluted samples were individuallyrun in a BioAnalyzer establishing the release of the oligonucleotidesinto the nanoreactors (FIG. 55).

Additional samples were taken as a positive control using a manualsyringe. Tygon tubing was used to create a face seal with theoligonucleotide synthesis device. The syringe, filled with 500 ul ofwater, was used to flush down liquid through one entire cluster as wellas parts of neighboring clusters from the handle side. The flushedliquid was collected in a 1.5 ml Eppendorf tube on the device side. Thesample was dried down in vacuum and then re-suspended in 10 uL water.The sample was then similarly analyzed in a BioAnalyzer. When accountingfor the dilution rates, a comparable concentration of oligonucleotideswere released using the positive control method and the nanoreactor blotmethod.

Example 12: Injection Based Oligonucleotide Transfer from anOligonucleotide Synthesis Device to a Nanoreactor

50-mer oligonucleotides are synthesized on a 3-D oligonucleotidesynthesis device as described in Example 9. The oligonucleotides arereleased from the surface by treatment in an ammonia chamber at about 75psi for about 14 hours. Alternatively, pressures from 20-120 psi can beused for 1-48 hours or longer for the release of the oligonucleotides.The temperature is room temperature. In some cases, the deprotectionrate may be increased by increasing the temperature, for example to atleast or at least about 25 C, 30 C, 35 C, 40 C, 45 C, 50 C, 55 C, 60 C,65 C or higher. Gaseous methylamine may also be used for deprotection atroom temperature or at an elevated temperature of at least or at leastabout 25 C, 30 C, 35 C, 40 C, 45 C, 50 C, 55 C, 60 C, 65 C or higher.The deprotection in methylamine typically proceeds faster than ingaseous ammonia.

The oligonucleotide synthesis device is assembled into a Hele Shaw flowcell with a single inlet and a single outlet. Flow is generated using asyringe that is connected to the flow cell via tygon tubing and ismanually controlled (FIG. 57). FIG. 56 illustrates a schematic of thefluidics in the flow cell. The fluidic circuit is used to flow fluidfrom the handle side into the first channels (or vias) and the fluid isfurther drawn into the second channels, e.g. those forming a revolverpattern comprising oligonucleotide synthesis sites. The fluid isdelivered from a single point inlet and collected from a single pointoutlet (FIG. 56 part B. In other cases, a line source and a line sinkcan be used to pass fluids (FIG. 56 part A). Without being bound bytheory, point source/sink combinations are expected to form a uniformair front, which can be more efficient to push all of the liquid outfrom the Hele-Shaw flow cell. Upon clearing of liquid from the flowcell, liquid is contained only in the vias on the handle side and thesecond channels or oligonucleotide synthesis channels, e.g. in arevolver pattern on the device side. This volume is estimated to be 300nL per cluster of vias (or first channels). Such containment of fluidcan facilitate the formation of uniform sessile droplets on the devicelayer surface of the oligonucleotide synthesis device.

For this step, a suitable release buffer, such as a PCA compatiblebuffer, is selected to dissolve the released oligonucleotides intosolution. Upon filling the vias and the second channels, the liquid isflushed out from the Hele-shaw flow cell on the handle surface of theoligonucleotide synthesis chip using about 500-1000 Pa, leaving liquidonly in the stagnant zone (handle and revolver) of the device, which isestimated to be 300 nL per assembly cluster (FIG. 56 part C). The singlepoint outlet is blocked and pressurized air is flown on the handle layersurface at about 3000-5000 Pa to eject droplets onto the device layersurface (FIG. 56 part D). Sufficient release buffer is pushed throughthe flow cell to form sessile drops emerging from the second channels(or oligonucleotide synthesis channels) onto the device side surface ofthe oligonucleotide synthesis device. The sessile drop size can be about300-400 nL, but can be varied to a suitable size according to theparticular dimensions of the oligonucleotide synthesis clusters and/orthe nanoreactors as well as according to the desired concentration ofthe oligonucleotides. For example, sessile drop sizes of about 500 nLcan be formed. The sessile drop formation is optionally monitored with amicroscope to make sure that the drop formation is complete across theoligonucleotide synthesis device. In some cases, the liquid forming thesessile drops may be prepared from a mixture of components so that adesired contact angle is achieved on the device layer. Accordingly, thesolution may be supplemented with a component, such as a detergent, e.g.polysorbate 20 (Polyoxyethylene (20) sorbitan monolaurate, akaTween-20).

Alternatively, a suitable amount of release buffer is deposited into theindividual wells/first channels from the handle side and pushed throughthe oligonucleotide synthesis channel, for example by applying pressurefrom the handle side by forming a Hele-shaw flow cell on the handleside. A nanoreactor device is mantled against the device side of theoligonucleotide synthesis device at a suitable rate, e.g. about 1-10mm/s and distance, e.g. about 50 um. The mantling can be performedquickly after drop formation to avoid evaporation. Evaporation is alsominimal once the two devices (nanoreactor and oligo synthesis reactor)are mantled.

Example 13: Gene Assembly in Nanoreactors Using PCA from ReactionMixtures Transferred from the Device Side of an OligonucleotideSynthesis

A PCA reaction mixture was prepared as described in Table 7 using theSEQ ID NOs.: 7-66 from Table 8, to assemble the 3075 bp LacZ gene (SEQID NO.: 67; Table 8).

TABLE 7 PCA 1 (×100 ul) final conc. H2O 62.00 5x Q5 buffer 20.00 1x 10mM dNTP 1.00 100 uM BSA 20 mg/ml 5.00 1 mg/ml Oligo mix 50 nM each 10.005 nM Q5 pol 2U/ul 2.00 2u/50 ul

TABLE 8 Sequence Name Sequence Oligo_1, SEQ ID NO.: 75′ATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAAC GTCGTGACTGGGAAAACCCTGG3′Oligo_2, SEQ ID NO.: 8 5′GCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGAC3′ Oligo_3, SEQ ID NO.: 95′CCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC3′ Oligo_4, SEQ ID NO.: 105′CGGCACCGCTTCTGGTGCCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGA3′ Oligo_5, SEQ ID NO.: 115′CACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTC3′ Oligo_6, SEQ ID NO.: 125′GATAGGTCACGTTGGTGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTTTGAGGGGACGACGACAGTATCGG3′ Oligo_7, SEQ ID NO.: 135′CCCATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTG3′ Oligo_8, SEQ ID NO.: 145′GTCTGGCCTTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAACCCGTCGGATTCTCCGTG3′ Oligo_9, SEQ ID NO.: 155′GCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGG3′ Oligo_10, SEQ ID NO.: 165′CAGGTCAAATTCAGACGGCAAACGACTGTCCTGGCCGTAACCGACCCAGCGCCCGTTGCACCACAGATGAAACG3′ Oligo_11, SEQ ID NO.: 175′CGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTG3′ Oligo_12, SEQ ID NO.: 185′GCCGCTCATCCGCCACATATCCTGATCTTCCAGATAACTGCCGTCACTCCAGCGCAGCACCATCACCGCGAG3′ Oligo_13, SEQ ID NO.: 195′AGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTC3′ Oligo_14, SEQ ID NO.: 205′CTCCAGTACAGCGCGGCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATTTGTGTAGTCGGTTTATG3′ Oligo_15, SEQ ID NO.: 215′ATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTT3′ Oligo_16, SEQ ID NO.: 225′AAAGGCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGCCATAAAGAAACTGTTACCCGTAGGTAGTCACG3′ Oligo_17, SEQ ID NO.: 235′GCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTTATGCCGATCGCGTCACACTACG3′ Oligo_18, SEQ ID NO.: 245′GATAGAGATTCGGGATTTCGGCGCTCCACAGTTTCGGGTTTTCGACGTTCAGACGTAGTGTGACGCGATCGGCA3′ Oligo_19, SEQ ID NO.: 255′GAGCGCCGAAATCCCGAATCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAG3′ Oligo_20, SEQ ID NO.: 265′CAGCAGCAGACCATTTTCAATCCGCACCTCGCGGAAACCGACATCGCAGGCTTCTGCTTCAATCAGCGTGCCG3′ Oligo_21, SEQ ID NO.: 275′CGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACGAGCATCA3′ Oligo_22, SEQ ID NO.: 285′GCAGGATATCCTGCACCATCGTCTGCTCATCCATGACCTGACCATGCAGAGGATGATGCTCGTGACGGTTAACGC3′ Oligo_23, SEQ ID NO.: 295′CAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAAC3′ Oligo_24, SEQ ID NO.: 305′TCCACCACATACAGGCCGTAGCGGTCGCACAGCGTGTACCACAGCGGATGGTTCGGATAATGCGAACAGCGCAC3′ Oligo_25, SEQ ID NO.: 315′GCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATG3′ Oligo_26, SEQ ID NO.: 325′GCACCATTCGCGTTACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGATCATCGGTCAGACGATTCATTGGCAC3′ Oligo_27, SEQ ID NO.: 335′CGCGTAACGCGAATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAG3′ Oligo_28, SEQ ID NO.: 345′GGATCGACAGATTTGATCCAGCGATACAGCGCGTCGTGATTAGCGCCGTGGCCTGATTCATTCCCCAGCGACCAGATG3′ Oligo_29, SEQ ID NO.: 355′GTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGTATGAAGGCGGCGGAGCCGACACCACGGC3′ Oligo_30, SEQ ID NO.: 365′CGGGAAGGGCTGGTCTTCATCCACGCGCGCGTACATCGGGCAAATAATATCGGTGGCCGTGGTGTCGGCTC3′ Oligo_31, SEQ ID NO.: 375′TGGATGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTACCTGGAGAGAC3′ Oligo_32, SEQ ID NO.: 385′CCAAGACTGTTACCCATCGCGTGGGCGTATTCGCAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGAAAGCC3′ Oligo_33, SEQ ID NO.: 395′CGCGATGGGTAACAGTCTTGGCGGTTTCGCTAAATACTGGCAGGCGTTTCGTCAGTATCCCCGTTTACAGGGC3′ Oligo_34, SEQ ID NO.: 405′GCCGTTTTCATCATATTTAATCAGCGACTGATCCACCCAGTCCCAGACGAAGCCGCCCTGTAAACGGGGATACTGACG3′ Oligo_35, SEQ ID NO.: 415′CAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACG3′ Oligo_36, SEQ ID NO.: 425′GCGGCGTGCGGTCGGCAAAGACCAGACCGTTCATACAGAA CTGGCGATCGTTCGGCGTATCGCCAAA3′Oligo_37, SEQ ID NO.: 43 5′CGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACACCAGCAGCAGTTTTTCCAGTTCCGTTTATCCG3′ Oligo_38, SEQ ID NO.: 445′CTCGTTATCGCTATGACGGAACAGGTATTCGCTGGTCACTTCGATGGTTTGCCCGGATAAACGGAACTGGAAAAACTGC3′ Oligo_39, SEQ ID NO.: 455′AATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATGGTGGCGCTGGATGGTAAGCCGCTGGCAAGCG3′ Oligo_40, SEQ ID NO.: 465′GTTCAGGCAGTTCAATCAACTGTTTACCTTGTGGAGCGACATCCAGAGGCACTTCACCGCTTGCCAGCGGCTTACC3′ Oligo_41, SEQ ID NO.: 475′CAAGGTAAACAGTTGATTGAACTGCCTGAACTACCGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACGCGTA3′ Oligo_42, SEQ ID NO.: 485′GCGCTGATGTGCCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGCACTACGCGTACTGTGAGCCAGAGTTG3′ Oligo_43, SEQ ID NO.: 495′CCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGA AAACCTCAGTGTGACGCTCCCCGCCGC3′Oligo_44, SEQ ID NO.: 50 5′CCAGCTCGATGCAAAAATCCATTTCGCTGGTGGTCAGATGCGGGATGGCGTGGGACGCGGCGGGGAGCGTC3′ Oligo_45, SEQ ID NO.: 515′CGAAATGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTG3′ Oligo_46, SEQ ID NO.: 525′TGAACTGATCGCGCAGCGGCGTCAGCAGTTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGCCTGACTGG3′ Oligo_47, SEQ ID NO.: 535′GCCGCTGCGCGATCAGTTCACCCGTGCACCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGAC3′ Oligo_48, SEQ ID NO.: 545′GGCCTGGTAATGGCCCGCCGCCTTCCAGCGTTCGACCCAGGCGTTAGGGTCAATGCGGGTCGCTTCACTTA3′ Oligo_49, SEQ ID NO.: 555′CGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCACGGCAGATACACTTGCTGATGCGGTGCTGAT3′ Oligo_50, SEQ ID NO.: 565′TCCGGCTGATAAATAAGGTTTTCCCCTGATGCTGCCACGCGTGAGCGGTCGTAATCAGCACCGCATCAGCAAGTG3′ Oligo_51, SEQ ID NO.: 575′GGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGGCGATTACCGTTGATGTTGA3′ Oligo_52, SEQ ID NO.: 585′GGCAGTTCAGGCCAATCCGCGCCGGATGCGGTGTATCGCTCGCCACTTCAACATCAACGGTAATCGCCATTTGAC3′ Oligo_53, SEQ ID NO.: 595′GCGGATTGGCCTGAACTGCCAGCTGGCGCAGGTAGCAGAGCGGGTAAACTGGCTCGGATTAGGGCCGCAAG3′ Oligo_54, SEQ ID NO.: 605′GGCAGATCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGTCGGGATAGTTTTCTTGCGGCCCTAATCCGAGC3′ Oligo_55, SEQ ID NO.: 615′GTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGC3′ Oligo_56, SEQ ID NO.: 625′GTCGCCGCGCCACTGGTGTGGGCCATAATTCAATTCGCGCGTCCCGCAGCGCAGACCGTTTTCGCTCGG3′ Oligo_57, SEQ ID NO.: 635′ACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATC3′ Oligo_58, SEQ ID NO.: 645′GAAACCGTCGATATTCAGCCATGTGCCTTCTTCCGCGTGCAGCAGATGGCGATGGCTGGTTTCCATCAGTTGCTG3′ Oligo_59, SEQ ID NO.: 655′CATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCG3′ Oligo_60, SEQ ID NO.: 665′TTATTTTTGACACCAGACCAACTGGTAATGGTAGCGACCGGCGCTCAGCTGGAATTCCGCCGATACTGACGGGC3′ LacZ gene-SEQ ID NO: 675′ATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGTCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCCATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTTATGCCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGATGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCGGCGATGAGCGAACGCGTAACGCGAATGGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGTATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTACCTGGAGAGACGCGCCCGCTGATCCTTTGCGAATACGCCCACGCGATGGGTAACAGTCTTGGCGGTTTCGCTAAATACTGGCAGGCGTTTCGTCAGTATCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACACCAGCAGCAGTTTTTCCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATGGTGGCGCTGGATGGTAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTCGCTCCACAAGGTAAACAGTTGATTGAACTGCCTGAACTACCGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACGCGTAGTGCAACCGAACGCGACCGCATGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGAAAACCTCAGTGTGACGCTCCCCGCCGCGTCCCACGCCATCCCGCATCTGACCACCAGCGAAATGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACAGATGTGGATTGGCGATAAAAAACAACTGCTGACGCCGCTGCGCGATCAGTTCACCCGTGCACCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCACGGCAGATACACTTGCTGATGCGGTGCTGATTACGACCGCTCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGGCGATTACCGTTGATGTTGAAGTGGCGAGCGATACACCGCATCCGGCGCGGATTGGCCTGAACTGCCAGCTGGCGCAGGTAGCAGAGCGGGTAAACTGGCTCGGATTAGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCCGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGT GTCAAAAATAA3′

Drops of about 400 nL were dispensed using a Mantis dispenser(Formulatrix, MA) on top of the revolvers (oligonucleotide synthesischannels) on the device side of an oligonucleotide synthesis device. Ananoreactor chip was manually mated with the oligonucleotide device topick up the droplets having the PCA reaction mixture. The droplets werepicked up into the individual nanoreactors in the nanoreactor chip byreleasing the nanoreactor from the oligonucleotide synthesis deviceimmediately after pick-up (FIG. 59).

The nanoreactors were sealed with a Heat Sealing Film/Tape cover(Eppendorf, eshop.eppendorfna.com/products/EppendorfHeat_Sealing_PCR_Film_and_Foil) and placed in a suitably configuredthermocycler that was constructed using a thermocycler kit (OpenPCR).

The following temperature protocol was used on the thermocycler:

1 cycle: 98 C, 45 seconds

40 cycles: 98 C, 15 seconds; 63 C, 45 seconds; 72 C, 60 seconds;

1 cycle: 72 C, 5 minutes

1 cycle: 4 C, hold

An aliquot of 0.50 ul was collected from individual wells 1-10 as shownin FIG. 60 and the aliquots were amplified in plastic tubes, in a PCRreaction mixture (Table 9) and according to the following thermocyclerprogram, using a forward (F-primer; 5′ATGACCATGATTACGGATTCACTGGCC3′; SEQID NO: 68) and a reverse (R-primer; 5′TTATTTTTGACACCAGACCAACTGGTAATGG3′;SEQ ID NO: 69) primer:

Thermocycler:

1 cycle: 98 C, 30 seconds

30 cycles: 98 C, 7 seconds; 63 C, 30 seconds; 72 C, 90 seconds

1 cycle: 72 C, 5 minutes

1 cycle: 4 C, hold

TABLE 9 PCR 1 (×25 ul) final conc. H2O 17.50 5x Q5 buffer 5.00 1x 10 mMdNTP 0.50 200 uM F-primer 20uM 0.63 0.5 uM R-primer 20uM 0.63 0.5 uM BSA20 mg/ml 0.00 Q5 pol 2U/ul 0.25 1u/50 ul template (PCA assembly) 0.50 1ul/50 ul rxn

The resulting amplification products were run on a BioAnalyzerinstrument (FIG. 60 part B, panels 1-10) as well as on a gel (FIG. 60part C), showing a product that is slightly larger than 3000 bp. An11^(th) PCR reaction was run using a PCA reaction performed in a plastictube as a positive control (FIG. 60 part B, panel 11 and FIG. 60 partC). A 12^(th) PCR reaction was run without the PCA template as anegative control showing no product (FIG. 60 part B, panel 12 and FIG.60 part C).

Example 14: Error Correction of Assembled Nucleic Acids

TABLE 10 Nucleic Acid Sequence Assembled Gene,5′ATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACT SEQ ID NO.: 70GGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGTCGTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCCATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTTATGCCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAACTGTGGAGCGCCGAAA TCCCGAATCTCTATC3′Assembly 5′ATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTOligonucleotide 1, GGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTSEQ ID NO.: 71 TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC 3′ Assembly5′GATAGGTCACGTTGGTGTAGATGGGCGCATCGTAACCGTGCATCTGCCA Oligonucleotide 2,GTTTGAGGGGACGACGACAGTATCGGCCTCAGGAAGATCGCACTCCAGCCA SEQ ID NO.: 72GCTTTCCGGCACCGCTTCTGGTGCCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGA3′ Assembly5′CCCATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTG Oligonucleotide 3,TTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATG SEQ ID NO.: 73AAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGG3′ Assembly5′GCCGCTCATCCGCCACATATCCTGATCTTCCAGATAACTGCCGTCACTC Oligonucleotide 4,CAGCGCAGCACCATCACCGCGAGGCGGTTTTCTCCGGCGCGTAAAAATGCG SEQ ID NO.: 74CTCAGGTCAAATTCAGACGGCAAACGACTGTCCTGGCCGTAACCGACCCAGCGCCCGTTGCACCACAGATGAAACG 3′ Assembly5′AGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGCTGCA Oligonucleotide 5,TAAACCGACTACACAAATCAGCGATTTCCATGTTGCCACTCGCTTTAATGA SEQ ID NO.: 75TGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTT 3′ Assembly5′GATAGAGATTCGGGATTTCGGCGCTCCACAGTTTCGGGTTTTCGACGTT Oligonucleotide 6,CAGACGTAGTGTGACGCGATCGGCATAACCACCACGCTCATCGATAATTTC SEQ ID NO.: 76ACCGCCGAAAGGCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGCCATAAAGAAACTGTTACCCGTAGGTAGTCACG 3′

An gene of about 1 kb (SEQ ID NO.: 70; Table 10) was assembled using 6purchased oligonucleotides (5 nM each during PCA) (Ultramer; SEQ ID NO.:71-76; Table 10) and assembled in a PCA reaction using a 1×NEB Q5 bufferwith 0.02 U/uL Q5 hot-start high-fidelity polymerase and 100 uM dNTP asfollows:

1 cycle: 98 C, 30 sec

15 cycles: 98 C, 7 sec; 62 C 30 sec; 72 C, 30 sec

1 cycle: 72 C, 5 min

Ultramer oligonucleotides are expected to have error rates of at least 1in 500 nucleotides, more likely at least 1 in 200 nucleotides or more.

The assembled gene was amplified in a PCR reaction using a forwardprimer (5′ ATGACCATGATTACGGATTCACTGGCC3′ SEQ ID NO.: 77) and a reverseprimer (5′GATAGAGATTCGGGATTTCGGCGCTCC

3′ SEQ ID NO.: 78), using 1×NEB Q5 buffer with 0.02 U/uL Q5 hot-starthigh-fidelity polymerase, 200 uM dNTP, and 0.5 uM primers as follows:

1 cycle: 98 C, 30 sec

30 cycles: 98 C, 7 sec; 65 C 30 sec; 72 C, 45 sec

1 cycle: 72 C, 5 min

The amplified assembled gene was analyzed in a BioAnalyzer (FIG. 52 partA) and cloned. Mini-preps from ˜24 colonies were Sanger sequenced. TheBioAnalyzer analysis provided a broad peak and a tail for theuncorrected gene, indicated a high error rate. The sequencing indicatedan error rate of 1/789 (data not shown). Two rounds of error correctionwere followed using CorrectASE (Life Technologies,www.lifetechnologies.com/order/catalog/product/A14972) according to themanufacturer's instructions. The resulting gene samples were similarlyanalyzed in the BioAnalyzer after round one (FIG. 60 part B) and roundtwo (FIG. 60 part C) and cloned. 24 colonies were picked for sequencing.The sequencing results indicated an error rate of 1/5190 bp and 1/6315bp after the first and second rounds of error correction, respectively.

Example 15: Generation of a Large Quantity of Primer-FreeSingle-Stranded Oligonucleotides

Reagents.

All enzymes and buffers except phi29 DNA polymerase were purchased fromNEB unless stated otherwise. Phi29 DNA polymerase was purchased fromEnzymatics.

Generation of Oligonucleotides.

A padlock oligonucleotide (OS_1518) having a reverse complement sequenceto a desired oligonucleotide was synthesized by IDT (Table 1).Additional padlock oligonucleotides OS_1515, OS_1516, OS_1517, OS_1519were also synthesized to work with adaptor/auxiliary oligonucleotidecombinations that work with different restriction enzyme sets. Thepadlock oligonucleotide was phosphorylated by mixing 5 μL padlock (200nM) with 5 μL T4 PNK buffer, 0.5 μL ATP (100 mM), 2 μL T4 PNK (10 U/μL),1 μL BSA (100 μg/μL), 2 μL DTT (100 mM), and 32.5 μL water, andincubating the mixture for 60 min at 37° C., followed by incubation for20 min at 65° C. An adaptor oligonucleotide having a complement sequenceto the padlock oligonucleotide was synthesized by IDT (Table 11). Anauxiliary oligonucleotide having a complementary sequence to the adaptoroligonucleotide was synthesized by IDT and biotinylated.

TABLE 11  Oligonucleotide sequences. Padlock, SEQ5′ATCTTTGAGTCTTCTGCTTGGTCAGACGAGTGCATGTGCGTGACAA ID NO.: 79ATTGGCGCGAGGAGCTCGTGTCATTCACAACTGCTCTTAGGCTACTCAGGCATGGTGAGATGCTACGGTGGTTGATGGATACCTAGAT3′ Adaptor, SEQ ID NO.: 80

Auxiliary, SEQ /5Biosg/GTTGATGGATACCTAGATATCTTTGAGTCTTCTG3′ ID NO.: 81Underline = complementarity to adaptor oligonucleotide Squigglyunderline = restriction site /5Biosg/ = biotinylation site

Hybridization and Ligation.

48 μL of the padlock phosphorylation reaction mixture was combined with1.5 μL adaptor oligonucleotide (2 μM) and 0.5 μL T4 ligase. The reactionwas incubated for 60 min at 37° C., followed by 20 min at 65° C. A 5 μLsample of the reaction was mixed with 5 μL 2× loading buffer andanalyzed on a 15% TBE-urea gel (180 V, 75 min).

An optional exonuclease treatment was performed as follows. A 10 μLligation product was treated with 0.15 μL ExoI and ExoIII (NEB orEnzymatics) at 37° C. for 60 min, followed by 95° C. for 20 min.Following incubation, 0.3 μL adaptor oligonucleotide (2 μM) was added toeach 10 μL solution, heated to 95° C. for 5 min, and slowly cooled. A 5μL sample of the reaction was mixed with 5 μL 2× loading buffer andanalyzed on a 15% TBE-urea gel (180 V, 75 min).

Rolling Circle Amplification.

A 10 μL 2×RCA master mix was prepared by combining 0.6 μL phi29 DNApolymerase (low concentration, Enzymatics), 0.5 μL 10 mM dNTP, 1 μL T4PNK buffer, 0.2 μL 100×BSA, 0.5 μL 100 mM DTT, and 7.2 μL water on ice.In some instances, PCR additives, such as betaine, for example 5Mbetaine, may be used to reduce amplification bias. The 10 μL of RCAmastermix was combined with 10 μL ligation product (with or withoutexonuclease treatment) and incubated at 40° C. for 90 min or 4 hr. Thereaction was then incubated at 70° C. for 10 min to de-activate thephi29 DNA polymerase. A 0.1 μL sample of the reaction was mixed with 4.9μL water and 5 μL 2× loading buffer, and the mixture analyzed on a 15%TBE-urea gel (180 V, 75 min).

Restriction Endonuclease Digestion.

A 2 μL sample of the RCA product was mixed with 2 μL 10× CutSmart, 2 μLbiotinylated auxiliary oligonucleotide (20 μM), and 12 μL water. Themixture was heated to 98° C. and slowly cooled to room temperature. 1 μLeach of BciVI and MlyI were added to the mixture, followed by anincubation for 1 hr at 37° C., then 20 min at 80° C. A 1 μL sample ofthe reaction was mixed with 4 μL water and 5 μL 2× loading buffer, andthe mixture analyzed on a 15% TBE-urea gel (180 V, 75 min).

An optional purification step was performed as follows. 1 μL of therestriction endonuclease digestion sample is retained as apre-purification sample. NanoLink beads (Solulink) are resuspended byvortexing vigorously. A 5 μL aliquot of beads were added to a 1.5 mLtube. Nucleic Acid Binding and Wash Buffer or NABWB (50 mM Tris-HCl, 150mM NaCl, 0.05% Tween 20, pH 8.0) was added to the tube to a final volumeof 250 μL, and the tube was mixed to resuspend. The tube was placed on amagnetic stand for 2 min, followed by removal of the supernatant. Thetube was removed from the magnet and the beads resuspended with 180 μLNABWB. 180 μL of the resuspended beads were added to 20 μL of therestriction endonuclease digestion reaction, and the mixture vortexed.The mixture was incubated for 60 min at 40° C. on a platform shaker, sothat the beads do not settle. The tube was then placed on a magnet for 2min and the supernatant comprising purified product was transferred to anew tube. A 10 μL sample of the purified product was mixed with 5 μL 2×loading buffer and analyzed on a 15% TBE-urea gel (180 V, 75 min). Theconcentration of the purified RCA product was measured using Qubit ssDNAkit.

Alternative Purification.

In some workflows, the digested oligonucleotides can be purified using(high-performance liquid chromatography) HPLC.

FIG. 63 depicts the separation of restriction enzyme cleavedamplification products, where each single stranded amplification producthas been hybridized with an auxiliary oligonucleotide complementary tothe amplification product at adaptor copy sites, prior to cleavage. Datarelating to the amplification of single stranded nucleic acids usingpadlock probes OS_1515, OS_1516, OS_1517, OS_1518, OS_1519, withdifferent sets of restriction enzymes are also shown.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The invention claimed is:
 1. A method for polynucleotide synthesis,comprising: a) providing instructions in a computer readablenon-transient medium for synthesis of preselected cDNA sequences; and b)synthesizing at least 20,000 polynucleotides encoding for thepreselected cDNA sequences, wherein each of the at least 20,000polynucleotides is at least 100 bases in length, wherein each of the atleast 20,000 polynucleotides comprises a first overlap region which iscomplementary to a second overlap region of another polynucleotide ofthe at least 20,000 polynucleotides such that a plurality of genes areformed when a subset of the at least 20,000 polynucleotides areassembled, wherein the first overlap region comprises at least 10 basesin length, and wherein the at least 20,000 polynucleotides encodesequences with an aggregate error rate of less than 1 in 1000 basescompared to the preselected cDNA sequences received in the instructionsprovided in the computer readable non-transient medium without errorcorrection.
 2. The method of claim 1, wherein at least 100,000polynucleotides are synthesized.
 3. The method of claim 1, wherein atleast 750 genes are formed when a subset of the at least 20,000polynucleotides are assembled.
 4. The method of claim 1, wherein atleast 10,000 genes are formed when a subset of the at least 20,000polynucleotides are assembled.
 5. The method of claim 1, wherein thefirst overlap region comprises a GC content of 35% to 65%.
 6. The methodof claim 1, wherein the first overlap region comprises 10 to 100 basesin length.
 7. The method of claim 1, wherein each polynucleotide is 100to 300 bases in length.
 8. The method of claim 1, wherein eachpolynucleotide is 100 to 200 bases in length.
 9. The method of claim 1,wherein each of the genes is at least 0.5 kb in length.
 10. The methodof claim 1, wherein each of the genes is at least 3 kb in length. 11.The method of claim 1, wherein the at least 20,000 polynucleotides aresynthesized on a structure.
 12. The method of claim 11, wherein thestructure is a solid support.
 13. The method of claim 1, furthercomprising hybridizing each polynucleotide in said subset to at leastanother polynucleotide in said subset to form the plurality of genes.